Phosphoramidates, phosphinic amides and related compounds and the use thereof to modulate the activity of endothelin

ABSTRACT

Phosphoramidates and phosphinic amides and related compounds are provided. Also provided are methods that use the compounds for modulating the activity of the endothelin family of peptides are provided. In particular, compounds having formula:                    
     in which A, B, R 1  and R 2  are as described are provided. Also provided are methods for treating endothelin-mediated disorders by administering effective amounts of one or more of these compounds or prodrugs thereof. Methods for elucidating the physiological and pathophysiological roles of endothelin, as well as isolating endothelin receptors are also provided.

RELATED APPLICATIONS

This application is a continuation of U.S application Ser. No.08/624,706, to Chan et al., filed Mar. 26, 1996, entitled“PHOSPHORAMIDATES, PHOSPHINIC AMIDES AND RELATED COMPOUNDS AND THE USETHEREOF TO MODULATE THE ACTIVITY OF ENDOTHELIN”now U.S. Pat. No.5,958,905. The above application is incorporated herein in its entirety.

FIELD OF THE INVENTION

The present invention relates to the compounds that modulate theactivity of the endothelin family of peptides. In particular,phosphoramidate compounds, phosphinic amide compounds and relatedcompounds and use of these compounds as endothelin antagonists areprovided.

BACKGROUND OF THE INVENTION

The vascular endothelium releases a variety of vasoactive substances,including the endothelium-derived vasoconstrictor peptide, endothelin(ET) (see, e.g., Vanhoutte et al. (1986) Annual Rev. Physiol. 48:307-320; Furchgott and Zawadski (1980) Nature 288: 373-376). Endothelin,which was originally identified in the culture supernatant of porcineaortic endothelial cells (see, Yanagisawa et al. (1 988) Nature 332:411-415), is a potent twenty-one amino acid peptide vasoconstrictor. Itis the most potent vasopressor known and is produced by numerous celltypes, including the cells of the endothelium, trachea, kidney andbrain. Endothelin is synthesized as a two hundred and three amino acidprecursor preproendothelin that contains a signal sequence which iscleaved by an endogenous protease to produce a thirty-eight (human) orthirty-nine (porcine) amino acid peptide. This intermediate, referred toas big endothelin, is processed in vivo to the mature biologicallyactive form by a putative endothelin-converting enzyme (ECE) thatappears to be a metal-dependent neutral protease (see, e.g., Kashiwabaraet al. (1989) FEBS Lttrs. 247: 337-340). Cleavage is required forinduction of physiological responses (see, e.g., von Geldern et al.(1991) Peptide Res. 4: 32-35). In porcine aortic endothelial cells, thethirty-nine amino acid intermediate, big endothelin, is hydrolyzed atthe Trp²¹-Val²² bond to generate endothelin-1 and a C-terminal fragment.A similar cleavage occurs in human cells from a thirty-eight amino acidintermediate. Three distinct endothelin isopeptides that exhibit potentvasoconstrictor activity have been identified. They are endothelin-1,endothelin-2 and endothelin-3.

The isopeptides are encoded by a family of three genes (see, e.g., Inoueet al. (1989) Proc. Natl. Acad. Sci. USA 86: 2863-2867; see, also Saidaet al. (1989) J. Biol. Chem. 264: 14613-14616). The nucleotide sequencesof the three human genes are highly conserved within the region encodingthe mature 21 amino acid peptides and the C-terminal portions of thepeptides are identical. Endothelin-2 is (Trp⁶,Leu⁷) endothelin-1 andendothelin-3 is (Thr²,Phe⁴,Thr⁵,Tyr⁶, Lys⁷,Tyr¹⁴) endothelin-1. Theendothelin peptides exhibit numerous biological activities in vitro andin vivo. Endothelin provokes a strong and sustained vasoconstriction invivo in rats and in isolated vascular smooth muscle preparations; italso provokes the release of eicosanoids and endothelium-derivedrelaxing factor (EDRF) from perfused vascular beds. Intravenousadministration of endothelin-1 and in vitro addition to vascular andother smooth muscle tissues produce long-lasting pressor effects andcontraction, respectively (see, e.g., Bolger et al. (1991) Can. J.Physiol. Pharmacol. 69: 406-413). In isolated vascular strips, forexample, endothelin-1 is a potent (EC₅₀=4×10⁻¹⁰ M), slow acting, butpersistent, contractile agent. A single dose in vivo elevates bloodpressure in about twenty to thirty minutes. Endothelin-inducedvasoconstriction is not affected by antagonists to knownneurotransmitters or hormonal factors, but is abolished by calciumchannel antagonists. The effect of calcium channel antagonists, however,is most likely the result of inhibition of calcium influx, since calciuminflux appears to be required for the long-lasting contractile responseto endothelin.

Endothelin also mediates renin release, stimulates ANP release andinduces a positive inotropic action in guinea pig atria. In the lung,endothelin-1 acts as a potent bronchoconstrictor (Maggi et al. (1989)Eur. J. Pharmacol. 160: 179-182). Endothelin increases renal vascularresistance, decreases renal blood flow, and decreases glomerularfiltrate rate. It is a potent mitogen for glomerular mesangial cells andinvokes the phosphoinoside cascade in such cells (Simonson et al. (1990)J. Clin. Invest. 85: 790-797). Release of endothelins from culturedendothelial cells is modulated by a variety of chemical and physicalstimuli and appears to be regulated at the level of transcription and/ortranslation. Expression of the gene encoding endothelin-1 is increasedby chemical stimuli, including adrenaline, thrombin and Ca²⁺ ionophore.The production and release of endothelin from the endothelium isstimulated by angiotensin II, vasopressin, endotoxin, cyclosporine andother factors (see, Brooks et al. (1991) Eur. J. Pharm. 194:115-117),and is inhibited by nitric oxide. Endothelial cells appear to secreteshort-lived endothelium-derived relaxing factors (EDRF), includingnitric oxide or a related substance (Palmer et al. (1987) Nature 327:524-526), when stimulated by vasoactive agents, such as acetylcholineand bradykinin. Endothelin-induced vasoconstriction is also attenuatedby atrial natriuretic peptide (ANP).

There are specific high affinity binding sites (dissociation constantsin the range of 2-6×10⁻¹⁰ M) for the endothelins in the vascular systemand in other tissues, including the intestine, heart, lungs, kidneys,spleen, adrenal glands and brain. Binding is not inhibited bycatecholamines, vasoactive peptides, neurotoxins or calcium channelantagonists. Endothelin binds and interacts with receptor sites that aredistinct from other autonomic receptors and voltage dependent calciumchannels. Competitive binding studies indicate that there are multipleclasses of receptors with different affinities for the endothelinisopeptides. The sarafotoxins, a group of peptide toxins from the venomof the snake Atractaspis eingadensis that cause severe coronaryvasospasm in snake bite victims, have structural and functional homologyto endothelin-1 and bind competitively to the same cardiac membranereceptors (Kloog et al. (1989) Trends Pharmacol. Sci. 10: 212-214).

Two distinct endothelin receptors, designated ET_(A) and ET_(B), havebeen identified and DNA clones encoding each receptor have been isolated(Arai et al. (1990) Nature 348: 730-732; Sakurai et al. (1990) Nature348: 732-735). Based on the amino acid sequences of the proteins encodedby the cloned DNA, it appears that each receptor contains seven membranespanning domains and exhibits structural similarity to G-protein-coupledmembrane proteins. Messenger RNA encoding both receptors has beendetected in a variety of tissues, including heart, lung, kidney andbrain. The distribution of receptor subtypes is tissue specific (Martinet al. (1989) Biochem. Biophys. Res. Commun. 162: 130-137). ET_(A)receptors appear to be selective for endothelin-1 and are predominant incardiovascular tissues. ET_(B) receptors are predominant innoncardiovascular tissues, including the central nervous system andkidney, and interact with the three endothelin isopeptides (Sakurai etal. (1990) Nature 348: 732-734). In addition, ET_(A) receptors occur onvascular smooth muscle, are linked to vasoconstriction and have beenassociated with cardiovascular, renal and central nervous systemdiseases; whereas ET_(B) receptors are located on the vascularendothelium, linked to vasodilation (Takayanagi et al. (1991) FEBSLttrs. 282: 103-106) and have been associated with bronchoconstrictivedisorders.

By virtue of the distribution of receptor types and the differentialaffinity of each isopeptide for each receptor type, the activity of theendothelin isopeptides varies in different tissues. For example,endothelin-1 inhibits ¹²⁵I-labelled endothelin-1 binding incardiovascular tissues forty to seven hundred times more potently thanendothelin-3. ¹²⁵I-labelled endothelin-1 binding innon-cardiovasculartissues, such as kidney, adrenal gland, andcerebellum, is inhibited to the same extent by endothelin-1 andendothelin-3, which indicates that ET_(A) receptors predominate incardiovascular tissues and ET_(B) receptors predominate innon-cardiovascular tissues.

Endothelin plasma levels are elevated in certain disease states (see,e.g., International PCT Application WO 94/27979, and U.S. Pat. No.5,382,569, which disclosures are herein incorporated in their entiretyby reference). Endothelin-1 plasma levels in healthy individuals, asmeasured by radioimmunoassay (RIA), are about 0.26-5 pg/ml. Blood levelsof endothelin-1 and its precursor, big endothelin, are elevated inshock, myocardial infarction, vasospastic angina, kidney failure and avariety of connective tissue disorders. In patients undergoinghemodialysis or kidney transplantation or suffering from cardiogenicshock, myocardial infarction or pulmonary hypertension levels as high as35 pg/ml have been observed (see, Stewart et al. (1991) Annals InternalMed. 114: 464-469). Because endothelin is likely to be a local, ratherthan a systemic, regulating factor, it is probable that the levels ofendothelin at the endothelium/smooth muscle interface are much higherthan circulating levels.

Elevated levels of endothelin have also been measured in patientssuffering from ischemic heart disease (Yasuda et al. (1990) Amer. HeartJ. 119:801-806, Ray et al. (1992) Br. Heart J. 67:383-386). Circulatingand tissue endothelin immunoreactivity is increased more than twofold inpatients with advanced atherosclerosis (Lerman et al. (1991) New Engl.J. Med. 325:997-1001). Increased endothelin immunoreactivity has alsobeen associated with Buerger's disease (Kanno et al. (1990) J. Amer.Med. Assoc. 264:2868) and Raynaud's phenomenon (Zamora et al. (1990)Lancet 336 1144-1147). Increased circulating endothelin levels wereobserved in patients who underwent percutaneous transluminal coronaryangioplasty (PTCA) (Tahara et al. (1991) Metab. Clin. Exp. 40:1235-1237;Sanjay et al. (1991) Circulation 84(Suppl. 4):726), and in individualswith pulmonary hypertension (Miyauchi et al. (1992) Jpn. J. Pharmacol.58:279P; Stewart et al. (1991) Ann. Internal Medicine 114:464-469).Thus, there is clinical human data supporting the correlation betweenincreased endothelin levels and numerous disease states.

Endothelin Agonists and Antagonists

Because endothelin is associated with certain disease states and isimplicated in numerous physiological effects, compounds that caninterfere with endothelin-associated activities, such asendothelin-receptor interaction and vasoconstrictor activity, are ofinterest. Compounds that exhibit endothelin antagonistic activity havebeen identified. For example, a fermentation product of Streptomycesmisakiensis, designated BE-18257B, has been identified as an ET_(A)receptor antagonist. BE-18257B is a cyclic pentapeptide,cyclo(D-Glu-L-Ala-allo-D-lle-L-Leu-D-Trp), which inhibits ¹²⁵I-labelledendothelin-1 binding in cardiovascular tissues in aconcentration-dependent manner (IC₅₀ 1.4 μM in aortic smooth muscle, 0.8μM in ventricle membranes and 0.5 μM in cultured aortic smooth musclecells), but fails to inhibit binding to receptors in tissues in whichET_(B) receptors predominate at concentrations up to 100 μM. Cyclicpentapeptides related to BE-18257B, such ascyclo(D-Asp-Pro-D-Val-Leu-D-Trp) (BQ-123), have been synthesized andshown to exhibit activity as ET_(A) receptor antagonists (see, U.S. Pat.No. 5,114,918 to Ishikawa et al.; see, also, EP A1 0 436 189 to BANYUPHARMACEUTICAL CO., LTD (Oct. 7, 1991)). Studies that measure theinhibition by these cyclic peptides of endothelin-1 binding toendothelin-specific receptors indicate that these cyclic peptides bindpreferentially to ET_(A) receptors. Other peptidic and non-peptidicET_(A) antagonists have been identified (see, e.g., U.S. Pat. Nos.5,352,800, 5,334,598, 5,352,659, 5,248,807, 5,240,910, 5,198,548,5,187,195, 5,082,838). These include other cyclic peptides,acyltripeptides, hexapeptide analogs, certain antraquinone derivatives,indanecarboxylic acids, certain N-pyrimidylbenzenesulfonamides, certainbenzenesulfonamides, and certain naphthalenesulfonamides (see, e.g.,Nakajima et al. (1991) J. Antibiot. 44:1348-1356; Miyata et al. (1992)J. Antibiot. 45:74-8; Ishikawa et al. (1992) J. Med. Chem. 35:2139-2142;U.S. Pat. No. 5,114,918 to Ishikawa et al.; EP A1 0 569 193; EP A1 0 558258; EP A1 0 436 189 to BANYU PHARMACEUTICAL CO., LTD (Oct. 7, 1991);Canadian Patent Application 2,067,288; Canadian Patent Application2,071,193; U.S. Pat. No. 5,208,243; U.S. Pat. No. 5,270,313; Cody et al.(1993) Med. Chem. Res. 3:154-162; Miyata et al. (1992) J. Antibiot45:1041-1046; Miyata et al. (1992) J. Antibiot 45:1029-1040, Fujimoto etal. (1992) FEBS Lett. 305:41-44; Oshashi et al. (1002) J. Antibiot45:1684-1685; EP A1 0 496 452; Clozel et al. (1993) Nature 365:759-761;International Patent Application WO93/08799; Nishikibe et al. (1993)Life Sci. 52:717-724; and Benigni et al. (1993) Kidney Int. 44:440-444,U.S. Pat. No. 5,464,853 and others). In general, the identifiedcompounds have activities in in vitro assays as ET_(A) antagonists atconcentrations on the order of about 50 μM-100 μM or less. A number ofsuch compounds have also been shown to possess activity in in vivoanimal models. Very few, if any, selective ET_(B) antagonists have beendescribed.

Certain phosphonic acid derivatives and related compounds have beenidentified that have activity as ECE inhibitors (see, eq, U.S. Pat. Nos.5,481,030, 5,476,847, 5,438,046, 5,380,921 and 5,330,978); there doesnot appear to be any evidence that any of these compounds inhibit theinteraction of an endothelin peptide with an endothelin receptor.

Endothelin Agonists and Antagonists as Therapeutic Agents

It has been recognized that compounds that exhibit activity at IC₅₀ orEC₅₀ concentrations on the order of 10⁻⁴M or lower in standard in vitroassays that assess endothelin antagonist or agonist activity havepharmacological utility (see, e.g., U.S. Pat. Nos. 5,352,800, 5,334,598,5,352,659, 5,248,807, 5,240,910, 5,198,548, 5,187,195, 5,082,838 and5,464,853). By virtue of this activity, such compounds are considered tobe useful for the treatment of hypertension such as peripheralcirculatory failure, heart disease such as angina pectoris,cardiomyopathy, arteriosclerosis, myocardial infarction, pulmonaryhypertension, vasospasm, vascular restenosis, Raynaud's disease,cerebral stroke such as cerebral arterial spasm, cerebral ischemia, latephase cerebral spasm after subarachnoid hemorrhage, asthma,bronchoconstriction, renal failure, particularly post-ischemic renalfailure, cyclosporine hephrotoxicity such as acute renal failure,colitis, as well as other inflammatory diseases, endotoxic shock causedby or associated with endothelin, and other diseases in which endothelinhas been implicated.

Thus, in view of the numerous physiological effects of endothelin andits association with certain diseases, endothelin is believed to play acritical role in these pathophysiological conditions (see, e.g., Saitoet al. (1990) Hypertension 15: 734-738; Tomita et al. (1989) N. Engl. J.Med. 321: 1127; Kurihara et al. (1989) J. Cardiovasc. Pharmacol.13(Suppl. 5): S13-S17; Doherty (1992) J. Med. Chem. 35: 1493-1508; Morelet al. (1989) Eur. J. Pharmacol. 167: 427-428). More detailed knowledgeof the function and structure of the endothelin peptide family shouldprovide insight into the progression and treatment of such conditions.

To aid in gaining further understanding of and to develop treatments forendothelin-mediated or related disorders, there is a need to identifycompounds that modulate or alter endothelin activity. Identification ofcompounds that modulate endothelin activity, such as compounds that actas specific antagonists or agonists, may not only aid in elucidating thefunction of endothelin, but may yield therapeutically useful compounds.In particular, compounds that specifically interfere with theinteraction of endothelin peptides with ET_(A), ET_(B) or otherreceptors should be useful in identifying essential characteristics ofendothelin peptides, should aid in the design of therapeutic agents, andmay be useful as disease specific therapeutic agents.

Therefore, it is an object herein to provide compounds that have theability to modulate the biological activity of one or more of theendothelin isopeptides. It is another object to provide compounds thathave use as endothelin antagonists. It is also an object to usecompounds that specifically interact with or inhibit the interaction ofendothelin peptides with endothelin receptors as therapeutic agents forthe treatment of endothelin-mediated diseases and disorders and/or asreagents for the identification of endothelin receptor subtypes and/orfor the elucidation of the physiological and pathophysiological roles ofendothelin.

SUMMARY OF THE INVENTION

Phosphoramidates, phosphinic amides and related compounds and methodsusing the compounds for modulating the interaction of an endothelinpeptide with endothelin receptors are provided. In particular,phosphoramidate compounds, phosphinic amide compounds and relatedcompounds, compositions containing these compounds and methods using thecompounds for inhibiting the binding of an endothelin peptide to ET_(A)and/or ET_(B) receptors are provided.

The methods are effected by contacting endothelin receptors with one ormore of the compounds prior to, simultaneously with, or subsequent tocontacting the receptors with an endothelin peptide.

The compounds provided herein have formula I:

in which Ar¹ is selected from among substituted and unsubstituted alkylgroups or aryl and heteroaryl groups containing one or more, preferablyone ring or two to three fused rings and from 3 up to about 21 membersin the ring(s), in which the heteroaryl groups contain one to threeheteroatoms selected from among O, S and N. In particular Ar¹ isselected from substituted or unsubstituted groups that include, but arenot limited to, the following: naphthyl, phenyl, biphenyl, quinolyl,thienyl, furyl, isoquinolyl, pyrrolyl, pyridyl, indolyl, oxadiazolyl,pyrazolyl, isoxazolyl, isothiazolyl, pyrimidyl, benzo[b]furyl,benzo[b]thienyl and other such aryl and heteroaryl groups. Preferredamong these groups are 5 to 6 membered aryl groups and heteroaryl groupsthat contain one or two heteroatom(s).

Ar₁ is unsubstituted or is substituted with one or more substituents,preferably selected from among NH₂, NO₂, halide, pseudohalide, alkyl,alkenyl, alkynyl, aryl, arylalkyl, heteroaryl, alkoxy, alkylamino,alkylthio, haloalkyl, alkylsulfinyl, alkylsulfonyl, aryloxy, arylamino,arylthio, arylsulfinyl, arylsulfonyl, haloaryl, alkoxycarbonyl,alkylcarbonyl, aminocarbonyl, arylcarbonyl, formyl, substituted orunsubstituted amido, substituted or unsubstituted ureido, in which thealkyl, alkenyl and alkynyl portions contain from 1 up to about 14 carbonatoms and are either straight or branched chains or cyclic, and the aryland heteroaryl portions contain from about 4 to about 16 members in thering.

Ar¹ is preferably a five or six membered aryl or heteroaryl ring havingat least two substituents selected independently from the groupconsisting of halide, pseudohalide, alkyl, alkoxy, alkenyl or alkynyland is more preferably selected from among thienyl, furyl, isoquinolyl,pyrrolyl, pyridyl, indolyl, oxadiazolyl, pyrazolyl, isoxazolyl,isothiazolyl and pyrimidyl.

A and B are any groups such that the resulting compound inhibits bindingby 50% of an endothelin peptide to an endothelin receptor, compared tobinding in the absence of the compound or in the absence of thereceptor, at a concentration of the compound of less than about 100 μM.

In particular, A and B are independently selected from among halo,alkyl, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl, alkoxy,thioalkoxy, alkylamino, alkylthio, haloalkyl, alkylsulfinyl,alkylsulfonyl, aryloxy, arylamino, arylthio, arylsulfinyl, arylsulfonyl,heteroaryloxy, heteroarylamino, heteroarylthio, haloaryl,alkoxycarbonyl, alkylcarbonyl, aminocarbonyl, arylcarbonyl, formyl,amido and ureido, in which the alkyl, alkenyl and alkynyl portionscontain from 1 up to about 14 carbon atoms, preferably about 1 to 6carbon atoms, and are either straight or branched chains or cyclic, andin which the aryl and heteroaryl portions are substituted orunsubstituted and contain from about 4 to about 16 members in the ring,preferably about 4 to 8 members. Preferably A and B are independentlyselected from among halo, alkyl, alkenyl, alkynyl, aryl, arylalkyl,heteroaryl, haloalkyl, aryloxy, arylamino, arylthio, heteroaryloxy,heteroarylamino, heteroarylthio and haloaryl.

A and B, in certain embodiments, are each independently selected fromalkyl, alkenyl, alkynyl, alkoxy, or A and B are respectively Ar²—X_(p)and Ar³—Y_(o) in which o and p are 0 or 1 and Ar² and Ar³ are eachindependently selected from aryl and heteroaryl, and particularly aryland heteroaryl groups that have 4 to 7 members, preferably 5 to 7members in the ring. In particular, Ar² and Ar³ are each independentlyselected from among phenyl, pyridyl, pyrimidyl, pyridazinyl, pyrrolyl,pyrazinyl, thienyl and furyl.

Ar₁ is, in certain embodiments, selected from groups such as:

where each R, which is the substituent or represents the substituent(s),which may be the same or different, on each group, is independentlyselected from NH₂, NO₂, halide, pseudohalide, alkyl, alkenyl, alkynyl,aryl, arylalkyl, heteroaryl, alkoxy, alkylamino, alkylthio, haloalkyl,alkylsulfinyl, alkylsulfonyl, aryloxy, arylamino, arylthio,arylsulfinyl, arylsulfonyl, haloaryl, alkoxycarbonyl, alkylcarbonyl,aminocarbonyl, arylcarbonyl, formyl, substituted or unsubstituted amidoand substituted or unsubstituted ureido, in which the alkyl, alkenyl andalkynyl portions contain from 1 up to about 14 carbon atoms and areeither straight or branched chains or cyclic, and the aryl andheteroaryl portions contain from about 4 to about 16 members in thering. R is preferably H, halide, pseudohalide, alkyl, alkoxy, alkenyl oralkynyl, and is most preferably H, lower alkyl, lower alkoxy or halide.

In preferred embodiments, Ar¹ an N-(5-isoxazolyl), N-(3-isoxazolyl) orN-pyridazinyl, preferably N-3-pyridazinyl, and A and B, independently,are aryl or aryloxy groups, and are preferably phenyl, phenoxy, loweralkyl, lower alkenyl or lower alkyoxy.

In the embodiments described in detail herein, Ar₁ is generally anisoxazolyl group and the compounds are represented by the formula II:

in which R¹ and R² are independently selected from H, NH₂, NO₂, halide,pseudohalide, alkyl, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl,alkoxy, alkylamino, alkylthio, alkylaryl, aryloxy, arylamino, arylthio,haloalkyl, haloaryl, formyl, substituted or unsubstituted amido andsubstituted or unsubstituted ureido, in which the alkyl, alkenyl andalkynyl portions contain from 1 up to about 14 carbon atoms and areeither straight or branched chains or cyclic, and the aryl andheteroaryl portions contain from about 4 to about 16 members, with theproviso that R² is not halide, pseudohalide, alkylsulfinyl,alkylsulfonyl, arylsulfinyl, arylsulfonyl, alkoxycarbonyl,alkylcarbonyl, aminocarbonyl or arylcarbonyl.

In the preferred compounds herein, R¹ and R² are independently selectedfrom among lower alkyl, lower alkenyl, lower alkynyl, lower haloalkyl,halide, pseudohalide or H; R² is not halide or pseudohalide.

In certain preferred embodiments described herein, A and B are aryl,aryloxy, arylthioxy, heteroaryl, heteroaryloxy or heteroarylthioxy thatpreferably contain 4 to 7 member, more preferably 5 or 6 members in eachring. Of interest herein are compounds in which A and B are 5- to6-membered aryloxy, aryl, heteroaryloxy, and heteroaryl groups. Ofparticular interest are compounds in which A and/or B are phenyl andphenoxy. In particular, in certain embodiments, A and B are eachindependently selected from alkyl, alkenyl, alkynyl alkoxy, or A and Bare respectively Ar²—X_(p) and Ar³—Y_(o) in which o and p are 0 or 1,and Ar² and Ar³ are each independently selected from aryl andheteroaryl, and particularly aryl and heteroaryl groups that have 4 to 7members, preferably 5 to 7 members in the ring. Ar² and Ar³ arepreferably each independently selected from among phenyl, pyridyl,pyrimidinyl, pyridazinyl, pyrrolyl, pyrazinyl, thienyl and furyl, andmore preferably phenyl, pyrimidinyl, pyrrolyl, thienyl and furyl, andmost preferably phenyl and pyrimidinyl.

Thus, among the compounds provided herein are compounds that haveformula III:

in which R¹ and R² are each independently selected from H, NO₂, N₃,halide, pseudohalide, alkyl, alkenyl, alkynyl, aryl, arylalkyl,heteroaryl, alkylaryl, alkoxy, alkylamino, alkylthio, alkylsulfinyl,alkylsulfonyl, aryloxy, arylalkyl, arylamino, arylthio, arylsulfinyl,arylsulfonyl, haloalkyl, haloaryl, alkoxycarbonyl, alkylcarbonyl,aminocarbonyl, arylcarbonyl, formyl, amido, ureido, in which the alkyl,alkenyl, alkynyl portions are straight or branched chains of from about1 up to about 10 carbons, preferably, 1 to about 5 or 6 carbons and inwhich the aryl portions contain from 3 up to about 10 carbons,preferably 6 carbons, with the proviso that R² is not halide,pseudohalide, alkylsulfinyl, alkylsulfonyl, arylsulfinyl, arylsulfonyl,alkoxycarbonyl, alkylcarbonyl, aminocarbonyl or arylcarbonyl.;

X and Y are each independently selected from among O, N, NR¹³, S or(CH₂)_(n) in which n is 0 to 10, preferably 0 to 3, more preferably 0 or1, most preferably 0;

o and p are independently 0 or 1;

R¹³ is hydrogen or a group containing up to about 30 carbon atoms,preferably 1 to 10, more preferably 1 to 6, selected from alkyl,alkenyl, alkynyl, aryl, alkylaryl, heterocyclyl, aralkyl, aralkoxy,cycloalkyl, cycloalkenyl, cycloalkynyl, C(O)R¹⁴ and S(O)_(n)R¹⁴ in whichn is 0-2; R¹⁴ is hydrogen, alkyl, alkenyl, alkynyl, aryl, alkylaryl,heterocyclyl, aralkyl, aralkoxy, cycloalkyl, cycloalkenyl orcycloalkynyl; R¹³ and R¹⁴ are unsubstituted or are substituted with oneor more substituents each selected independently from Z, which ishydrogen, halide, pseudohalide, alkyl, alkoxy, alkenyl, alkynyl, aryl,heterocyclyl, aralkyl, aralkoxy, cycloalkyl, cycloalkenyl, cycloalkynyl,and preferably selected from among H, a straight or branched carbonchain, preferably containing 1 to 6, more preferably 1 to 3, carbons,halide, alkoxyalkyl, or haloalkyl; and

R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹ and R¹² are each independentlyselected from among any of (i), (ii), (iii), (iv) or (v):

(i) R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹ and R¹² are each independentlyselected from among H, NHOH, NH₂, NO₂, N₃, halide, pseudohalide, alkyl,alkenyl, alkynyl, aryl, arylalkyl, heteroaryl, alkoxy, alkylamino,alkylthio, alkoxyalkyl, alkylsulfinyl, alkylsulfonyl, aryloxy,arylamino, arylthio, arylalkyl, alkylaryl, arylsulfinyl, arylsulfonyl,haloalkyl, haloaryl, alkoxycarbonyl, alkylcarbonyl, aminocarbonyl,arylcarbonyl, formyl, amido, ureido, or at least two of R³, R⁴, R⁵, R⁶and R⁷ or R⁸, R⁹, R¹⁰, R¹¹ and R¹² form alkylenedioxy, in which thealkyl, alkenyl, alkynyl portions are straight or branched chains of fromabout 1 up to about 10 carbons, preferably 1 to about 5 or 6 carbons andin which the aryl portions contain from 3 up to about 10 carbons,preferably 6 carbons; or

(ii) R⁴ and R⁷ and/or R¹⁰ and R¹¹ together are substituted orunsubstituted 1,3-butadienyl, 4-dimethylamino-1,3-butadienyl,1-chloro-1,3-butadienyl, 1-aza-1,3-butadienyl or 2-aza-1,3-butadienylgroups; and the others of R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹ and R¹²are as defined in (i) above; or alternatively,

(iii) R⁷ and R³ and/or R¹¹ and R¹² together are substituted orunsubstituted 1,3-butadienyl, 4-dimethylamino-1,3-butadienyl,1-chloro-1,3-butadienyl, 1-aza-1,3-butadienyl or 2-aza-1,3-butadienylgroups; and the others of R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹ and R¹²are as defined in (i) above; or

(iv) R³, R⁵, and R⁷ and R¹⁰, R¹¹ and R⁸ are H or are as defined in (i);and R⁴ and R⁶ and/or R⁹ and R¹² are each independently selected fromalkyl, alkoxy, halide, aminoalkyl, dialkylaminoalkyl, in which the alkyland alkoxy groups contain from 1 to 10, preferably 1 to 6 carbons, andare straight or branched chains, and the others of R³, R⁴, R⁵, R⁶, R⁷,R⁸, R⁹, R¹⁰, R¹¹ and R¹² are as defined in (i) above; or

(v) any two of R³, R⁴, R⁵, R⁶, and R⁷, and/or any two of R⁸, R⁹, R¹⁰,R¹¹ and R¹², which are each selected as in (i) form fused carbocyclic orheterocyclic rings.

In particularly preferred embodiments described herein, the compoundshave formula III, in which R¹ and R² are independently selected fromamong lower alkyl, lower alkenyl, lower alkynyl, lower haloalkyl,halide, pseudohalide or H, with the proviso that R² is not halide orpseudohalide.

In the preferred compounds, X and Y are independently selected fromamong (CH₂)_(n) in which n is 1 to 3, and 0;

p and o are 0 or 1; and

R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹ and R¹² are independently selectedfrom among H, NH₂, halide, pseudohalide, lower alkyl, lower alkenyl andlower haloalkyl and at least three of R³, R⁴, R⁵, R⁶ and R⁷ are hydrogenand at least three of R⁸, R⁹, R¹⁰, R¹¹ and R¹² are hydrogen.

In more preferred compounds, R² is selected from among lower alkyl,lower alkenyl, lower alkynyl, lower haloalkyl; and R¹ is lower alkyl orhalide, particularly bromide or chloride.

Of the compounds described herein, those that inhibit or increase anendothelin-mediated activity by about 50% at concentrations of less thanabout 10 μM are preferred. More preferred are those that inhibit orincrease an endothelin-mediated activity by about 50% at concentrationsof less than about 5 μM and most preferably less than about 1 μM. Thepreferred IC₅₀ concentrations are set forth with reference to the invitro assays exemplified herein.

Also among the most preferred compounds, for use in the methods providedherein, are those that are ET_(A) or ET_(B) selective. A compound isconsidered selective for a particular endothelin receptor subtype if itinhibits endothelin binding to the receptor at an IC₅₀ concentrationthat is at least 10-fold lower than its IC₅₀ concentration for otherendothelin receptor subtypes. In particular, compounds that interactwith ET_(A) receptors with an lC₅₀ of less than about 10 μM, preferablyless than 1 μM, but with ET_(B) receptors with an IC₅₀ of greater thanabout 10 μM or compounds that interact with ET_(B) receptors with anIC₅₀ of less than about 10 μM, preferably less than 1 μM, but withET_(A) receptors with an IC₅₀ of greater than about 10 μM are preferred.

Of particular interest herein are compounds that inhibit binding ofendothelin to ET_(A) receptors at lower concentrations, preferably atleast 5- to 10-fold, more preferably at least 50-fold and mostpreferably 100-fold, lower than they inhibit binding to ET_(B)receptors.

Preferred compounds are ET_(A) receptor selective or bind to ET_(A)receptors with an IC₅₀ of less than about 20 μM, more preferably lessthan about 10 μM and most preferably less than about 5 μM.

Preferred compounds also include compounds that are ET_(B) receptorselective or that competitively inhibit binding of endothelin-1 toET_(B) receptors at IC₅₀ concentrations of less than about 20 μM.

Pharmaceutical compositions formulated for administration by anappropriate route and by appropriate means, containing effectiveconcentrations of one or more of the compounds provided herein orpharmaceutically acceptable salts or esters thereof, that deliveramounts effective for the treatment of hypertension, stroke, asthma,shock, ocular hypertension, glaucoma, renal failure, inadequate retinalperfusion or other conditions that are in some manner mediated by anendothelin peptide or that involve vasoconstriction or whose symptomscan be ameliorated by administration of an endothelin antagonist oragonist, are also provided. Particularly preferred compositions arethose that deliver amounts effective for the treatment of hypertensionor renal failure. The effective amounts and concentrations are effectivefor ameliorating any of the symptoms of any of the disorders.

Methods for treatment of endothelin-mediated disorders, including butnot limited to, hypertension, asthma, shock, ocular hypertension,glaucoma, inadequate retinal perfusion and other conditions that are insome manner mediated by an endothelin peptide, or for treatment ofdisorders that involve vasoconstriction or that are ameliorated byadministration of an endothelin antagonist or agonist are provided.

In particular, methods of treating endothelin-mediated disorders byadministering effective amounts of the compounds, prodrugs or othersuitable derivatives of the compounds are provided. Such disordersinclude but are not limited to hypertension, cardiovascular diseases,cardiac diseases including myocardial infarction, pulmonaryhypertension, erythropoietin-mediated hypertension, respiratory diseasesand inflammatory diseases, including asthma, bronchoconstriction,ophthalmologic diseases, gastroenteric diseases, renal failure,endotoxin shock, menstrual disorders, obstetric conditions, wounds,anaphylactic shock, hemorrhagic shock, and other diseases in whichendothelin mediated physiological responses are implicated. Treatmentinvolves, for instance, administration of effective amounts of one ormore of the compounds provided herein in pharmaceutically acceptablecarriers as provided herein. Preferred methods of treatment are methodsfor treatment of hypertension and renal failure.

More preferred methods of treatment are those in which the compositionscontain at least one compound that inhibits the interaction ofendothelin-1 with ET_(A) or ET_(B) receptors at an IC₅₀ of less thanabout 10 M, preferably less than about 5 μM and more preferably lessthan about 1 μM. Other preferred methods are those in which thecompositions contain one or more compounds that is (are) ET_(A)selective or one or more compounds that is (are) ET_(B) selective.Methods in which the compounds are ET_(A) selective are for treatment ofdisorders, such as hypertension; and methods in which the compounds areET_(B) selective are for treatment of disorders, such as asthma, thatrequire bronchodilation.

In practicing the methods, effective amounts of compositions containingtherapeutically effective concentrations of the compounds formulated fororal, intravenous, local and topical application for the treatment ofhypertension, cardiovascular diseases, cardiac diseases, includingmyocardial infarction, respiratory diseases, including asthma,inflammatory diseases, ophthalmologic diseases, gastroenteric diseases,renal failure, immunosuppressant-mediated renal vasoconstriction,erythropoietin-mediated vasoconstriction, endotoxin shock, anaphylacticshock, hemorrhagic shock, pulmonary hypertension, and other diseases inwhich endothelin mediated physiological responses are implicated areadministered to an individual exhibiting the symptoms of one or more ofthese disorders. The amounts are effective to ameliorate or eliminateone or more symptoms of the disorders.

Methods for the identification and isolation of endothelin receptorsubtypes are also provided. In particular, methods for detecting,distinguishing and isolating endothelin receptors using the disclosedcompounds are provided.

In addition, methods for identifying compounds that are suitable for usein treating particular diseases based on their preferential affinity fora particular endothelin receptor subtype are provided.

Also provided are methods for elucidating the physiological and/orpathophysiological roles of endothelin using the compounds disclosedherein.

Articles of manufacture containing packaging material, a compoundprovided herein, which is effective for ameliorating the symptoms of anendothelin-mediated disorder, antagonizing the effects of endothelin orinhibiting binding of an endothelin peptide to an ET receptor with anIC₅₀ of less than about 50 μM, preferably 10 μM, within the packagingmaterial, and a label that indicates that the compound or salt thereofis used for antagonizing the effects of endothelin, treating anendothelin-mediated disorder, or inhibiting the binding of an endothelinpeptide to an ET receptor are provided.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which this invention belongs. All patents and publicationsreferred to herein are incorporated by reference.

As used herein, endothelin (ET) peptides include peptides that havesubstantially the amino acid sequence of endothelin-1, endothelin-2 orendothelin-3 and that act as potent endogenous vasoconstrictor peptides.

As used herein, an endothelin-mediated condition is a condition that iscaused by abnormal endothelin activity or one in which compounds thatinhibit endothelin activity have therapeutic use. Such diseases include,but are not limited to hypertension, cardiovascular disease, asthma,inflammatory diseases, ophthalmologic disease, menstrual disorders,obstetric conditions, gastroenteric disease, renal failure, pulmonaryhypertension, endotoxin shock, anaphylactic shock, or hemorrhagic shock.Endothelin-mediated conditions also include conditions that result fromtherapy with agents, such as erythropoietin and immunosuppressants, thatelevate endothelin levels.

As used herein an effective amount of a compound for treating aparticular disease is an amount that is sufficient to ameliorate, or insome manner reduce the symptoms associated with the disease. Such amountmay be administered as a single dosage or may be administered accordingto a regimen, whereby it is effective. The amount may cure the diseasebut, typically, is administered in order to ameliorate the symptoms ofthe disease. Typically, repeated administration is required to achievethe desired amelioration of symptoms.

As used herein, an endothelin agonist is a compound that potentiates orexhibits a biological activity associated with or possessed by anendothelin peptide.

As used herein, an endothelin antagonist is a compound, such as a drugor an antibody, that inhibits endothelin-stimulated vasoconstriction andcontraction and other endothelin-mediated physiological responses. Theantagonist may act by interfering with the interaction of the endothelinwith an endothelin-specific receptor or by interfering with thephysiological response to or bioactivity of an endothelin isopeptide,such as vasoconstriction. Thus, as used herein, an endothelin antagonistinterferes with endothelin-stimulated vasoconstriction or other responseor interferes with the interaction of an endothelin with anendothelin-specific receptor, such as ET_(A) receptors, as assessed byassays known to those of skill in the art.

The effectiveness of potential agonists and antagonists can be assessedusing methods known to those of skill in the art. For example,endothelin agonist activity can be identified by its ability tostimulate vasoconstriction of isolated rat thoracic aorta or portal veinring segments (Borges et al. (1989) “Tissue selectivity of endothelin ”Eur. J. Pharmacol. 165: 223-230). Exemplary assays are set forth in theEXAMPLES. As noted above, the preferred IC₅₀ concentration ranges areset forth with reference to assays in which the test compound isincubated with the ET receptor-bearing cells at 4° C. Data presented forassays in which the incubation step is performed at the less preferred24° C. are identified. It is understood that for purposes of comparison,these concentrations are somewhat higher than the concentrationsdetermined at 4° C.

As used herein, the biological activity or bioactivity of endothelinincludes any activity induced, potentiated or influenced by endothelinin vivo. It also includes the ability to bind to particular receptorsand to induce a functional response, such as vasoconstriction. It may beassessed by in vivo assays or by in vitro assays, such as thoseexemplified herein. The relevant activities include, but are not limitedto, vasoconstriction, vasorelaxation and bronchodilation. For example,ET_(B) receptors appear to be expressed in vascular endothelial cellsand may mediate vasodilation and other such responses; whereas ET_(A)receptors, which are endothelin-1-specific, occur on smooth muscle andare linked to vasoconstriction Any assay known to those of skill in theart to measure or detect such activity may be used to assess suchactivity (see, e.g., Spokes et al. (1989) J. Cardiovasc. Pharmacol.13(Suppl. 5):S191-S192; Spinella et al. (1991) Proc. Natl. Acad. Sci.USA 88: 7443-7446; Cardell et al. (1991) Neurochem. Int. 18:571-574);and the Examples herein).

As used herein, the IC₅₀ refers to an amount, concentration or dosage ofa particular test compound that achieves a 50% inhibition of a maximalresponse, such as binding of endothelin to tissue receptors, in an assaythat measures such response.

As used herein, EC₅₀ refers to a dosage, concentration or amount of aparticular test compound that elicits a dose-dependent response at 50%of maximal expression of a particular response that is induced, provokedor potentiated by the particular test compound.

As used herein a phosphoramidate and/or phosphinic amide or relatedcompounds that is ET_(A) selective refers to one that exhibits an IC₅₀that is at least about 10-fold lower with respect to ET_(A) receptorsthan ET_(B) receptors.

As used herein, a phosphoramidate and/or phosphinic amide or relatedcompound that is ET_(B) selective refers to one that exhibits an IC₅₀that is at least about 10-fold lower with respect to ET_(B) receptorsthan ET_(A) receptors.

As used herein, a phosphoramidate refers to compounds, such as those ofcompound of formula III in which o and p are 1; a phosphinic amiderefers to compounds in which o and p are 0, and related compounds referto compounds in which one of o and p is 0 and the other is 1.

As used herein, pharmaceutically acceptable salts, esters or otherderivatives of the compounds include any salts, esters or derivativesthat may be readily prepared by those of skill in this art using knownmethods for such derivatization and that produce compounds that may beadministered to animals or humans without substantial toxic effects andthat either are pharmaceutically active or are prodrugs. For example,hydroxy groups can be esterified or etherified.

As used herein, treatment means any manner in which the symptoms of aconditions, disorder or disease are ameliorated or otherwisebeneficially altered. Treatment also encompasses any pharmaceutical useof the compositions herein, such as use as contraceptive agents.

As used herein, amelioration of the symptoms of a particular disorder byadministration of a particular pharmaceutical composition refers to anylessening, whether permanent or temporary, lasting or transient that canbe attributed to or associated with administration of the composition.

As used herein, substantially pure means sufficiently homogeneous toappear free of readily detectable impurities as determined by standardmethods of analysis, such as thin layer chromatography (TLC), gelelectrophoresis and high performance liquid chromatography (HPLC), usedby those of skill in the art to assess such purity, or sufficiently puresuch that further purification would not detectably alter the physicaland chemical properties, such as enzymatic and biological activities, ofthe substance. Methods for purification of the compounds to producesubstantially chemically pure compounds are known to those of skill inthe art. A substantially chemically pure compound may, however, be amixture of stereoisomers. In such instances, further purification mightincrease the specific activity of the compound.

As used herein, biological activity refers to the in vivo activities ofa compound or physiological responses that result upon in vivoadministration of a compound, composition or other mixture. Biologicalactivity, thus, encompasses therapeutic effects and pharmaceuticalactivity of such compounds, compositions and mixtures.

As used herein, a prodrug is a compound that, upon in vivoadministration, is metabolized or otherwise converted to thebiologically, pharmaceutically or therapeutically active form of thecompound. To produce a prodrug, the pharmaceutically active compound ismodified such that the active compound will be regenerated by metabolicprocesses. The prodrug may be designed to alter the metabolic stabilityor the transport characteristics of a drug, to mask side effects ortoxicity, to improve the flavor of a drug or to alter othercharacteristics or properties of a drug. By virtue of knowledge ofpharmacodynamic processes and drug metabolism in vivo, those of skill inthis art, once a pharmaceutically active compound is known, can designprodrugs of the compound (see, e.g., Nogrady (1985) Medicinal ChemistryA Biochemical Approach, Oxford University Press, New York, pages388-392). For example, succinyl-sulfathiazole is a prodrug of4-amino-N-(2-thiazoyl)benzenesulfonamide (sulfathiazole) that exhibitsaltered transport characteristics.

As used herein, “halogen” or “halide” refers to F, Cl, Br or I.

As used herein, pseudohalides are compounds that behave substantiallysimilar to halides. Such compounds can be used in the same manner andtreated in the same manner as halides (X⁻, in which X is a halogen, suchas Cl or Br). Pseudohalides include, but are not limited to cyanide,cyanate, thiocyanate, selenocyanate and azide.

As used herein, lower alkyl, lower alkenyl, and lower alkynyl refer tocarbon chains having less than about 6 carbons. In preferred embodimentsof the compounds provided herein that include alkyl, alkenyl, or alkynylportions include lower alkyl, lower alkenyl, and lower alkynyl portions.

As used herein aryl refers to cyclic groups containing from 3 to 15 or16 carbon atoms, preferably from 5 to 10. Aryl groups include, but arenot limited to groups, such as phenyl, substituted phenyl, napthyl,substituted naphthyl, in which the substituent is lower alkyl, halide,O, or lower alkoxy. Preferred aryl groups are lower aryl groups thatcontain less than 7 carbons in the ring structure.

As used herein, the nomenclature alkyl, alkoxy, carbonyl, etc. are usedas is generally understood by those of skill in this art. For example,as used herein alkyl refers to saturated carbon chains that contain oneor more carbons; the chains may be straight or branched or includecyclic portions or may be cyclic.

As used herein, “haloalkyl” refers to a lower alkyl radical in which oneor more of the hydrogen atoms are replaced by halogen including, but notlimited to, chloromethyl, trifluoromethyl, 1-chloro-2-fluoroethyl andthe like.

As used herein, “haloalkoxy” refers to RO— in which R is a haloalkylgroup.

As used herein, “aminocarbonyl” refers to —C(O)NH₂.

As used herein, “alkoxycarbonyl” refers to —C(O)OR in which R is alkyl,preferably lower alkyl or aryl, preferably lower aryl.

As used herein, “alkoxy” and “thioalkoxy” refer to RO— and RS—, in whichR is alkyl, preferably lower alkyl or aryl, preferably lower aryl.

As used herein, “haloalkoxy” refers to RO— in which R is a haloalkylgroup.

As used herein, heteroaryl refers to ring structures that include atleast one carbon atom and at least one non-carbon atom, such as N, S, Pand O. The rings may be single rings or two or more fused rings.

As used herein, any corresponding N-(4-halo-3-methyl-5-isoxazolyl),N-(4-halo-5-methyl-3-isoxazolyl), N-(3,4-dimethyl-5-isoxazolyl),N-(4-halo-5-methyl-3-isoxazolyl), N-(4-halo-3-methyl-5-isoxazolyl),N-(4,5-dimethyl-3-isoxazolyl) derivative thereof refers to compounds inwhich A, B, X and Y are the same as the compound specifically set forth,but Ar₁ is N-(4-halo-3-methyl-5-isoxazolyl),N-(4-halo-5-methyl-3-isoxazolyl), N-(3,4-dimethyl-5-isoxazolyl),N-(4-halo-5-methyl-3-isoxazolyl), N-(4-halo-3-methyl-5-isoxazolyl), orN-(4,5-dimethyl-3-isoxazolyl) in which halo is any halide, preferably Clor Br.

As used herein, the abbreviations for any protective groups, amino acidsand other compounds, are, unless indicated otherwise, in accord withtheir common usage, recognized abbreviations, or the IUPAC-IUBCommission on Biochemical Nomenclature (see, (1972) Biochem.11:942-944).

A. Compounds for Use in Treating Endothelin-mediated Diseases

Compounds and methods for treating endothelin-mediated diseases usingthe compounds of formula I are provided. In particular, among thecompounds provided herein are those in which Ar¹ is an isoxazolyl groupand the compounds are represented by the formula II:

in which R¹ and R² are independently selected from H, NH₂, NO₂, halide,pseudohalide, alkyl, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl,alkoxy, alkylamino, alkylthio, alkylsulfinyl, alkylsulfonyl, aryloxy,arylamino, arylthio, arylsulfinyl, arylsulfonyl, haloalkyl, haloaryl,alkoxycarbonyl, alkylcarbonyl, aminocarbonyl, arylcarbonyl, formyl,substituted or unsubstituted amido, substituted or unsubstituted ureido,in which the alkyl, alkenyl and alkynyl portions contain from 1 up toabout 14 carbon atoms and are either straight or branched chains orcyclic, and the aryl and heteroaryl portions contain from about 4 toabout 16 members, with the proviso that R² is not halide, pseudohalide,alkylsulfinyl, alkylsulfonyl, arylsulfinyl, arylsulfonyl,alkoxycarbonyl, alkylcarbonyl, aminocarbonyl or arylcarbonyl.

In the preferred compounds herein, R¹ and R² are independently selectedfrom among lower alkyl, lower alkenyl, lower alkynyl, lower haloalkyl,halide, pseudohalide or H; R²is not halide or pseudohalide.

In certain preferred embodiments described herein, A and B are aryl,aryloxy, arylthioxy, heteroaryl, heteroaryloxy or heteroarylthioxy thatpreferably contain 4 to 7 members, more preferably 5 or 6 members ineach ring. Of interest herein are compounds in which A and B are 5 tosix- membered aryloxy, aryl, heteroaryloxy, and heteroaryl groups. Ofparticular interest are compounds in which A and/or B are phenyl andphenoxy.

In particular, in certain embodiments, A and B are each independentlyselected from alkyl, alkenyl, alkynyl, alkoxy, or A and B arerespectively Ar²—X_(p) and Ar³—Y_(o) in which o and p are 0 or 1, andAr² and Ar³ are each independently selected from aryl and heteroaryl,and particularly aryl and heteroaryl groups that have 4 to 7 members,preferably 5 to 7 members in the ring. Ar² and Ar³ are preferably eachindependently selected from among phenyl, pyridyl, pyrimidyl,pyridazinyl, pyrrolyl, pyrazinyl, thienyl and furyl, and more preferablyphenyl, pyrimidyl, pyrrolyl, thienyl and furyl, and most preferablyphenyl and pyrimidyl.

Of interest herein are compounds in which Ar² and Ar³ are 5- to6-membered aryl and heteroaryl groups. Of particular interest arecompounds in which A and/or B are phenyl and phenoxy.

Thus, among the compounds provided herein are compounds that haveformula III:

in which R¹ and R² are each independently selected from H, NHOH, NH₂,NO₂, N₃, halide, pseudohalide, alkyl, alkenyl, alkynyl, aryl, arylalkyl,heteroaryl, alkoxy, alkylamino, alkylthio, alkylsulfinyl, alkylsulfonyl,aryloxy, arylamino, arylthio, arylsulfinyl, arylsulfonyl, haloalkyl,haloaryl, alkoxycarbonyl, alkylcarbonyl, aminocarbonyl, arylcarbonyl,formyl, amido, ureido, in which the alkyl, alkenyl, alkynyl portions arestraight or branched chains of from about 1 up to about 10 carbons,preferably, 1 to about 5 or 6 carbons and in which the aryl portionscontain from 3 up to about 10 carbons, preferably 6 carbons;

X and Y are each independently selected from among O, N, NR¹³, S or(CH₂)_(n) in which n is 1 to 10, preferably 1 to 3, more preferably 1;

o and p are independently 0 or 1;

R¹³ is hydrogen or a group containing up to about 30 carbon atoms,preferably 1 to 10, more preferably 1 to 6, selected from alkyl,alkenyl, alkynyl, aryl, alkylaryl, heterocyclyl, aralkyl, aralkoxy,cycloalkyl, cycloalkenyl, cycloalkynyl, C(O)R¹⁴ and S(O)_(n)R¹⁴ in whichn is 0-2; R¹⁴ is hydrogen, alkyl, alkenyl, alkynyl, aryl, alkylaryl,heterocyclyl, aralkyl, aralkoxy, cycloalkyl, cycloalkenyl orcycloalkynyl; R¹³ and R¹⁴ are unsubstituted or are substituted with oneor more substituents each selected independently from Z, which ishydrogen, halide, pseudohalide, a alkyl, alkoxy, alkenyl, alkynyl, aryl,heterocyclyl, aralkyl, aralkoxy, cycloalkyl, cycloalkenyl, cycloalkynyl,and preferably selected from among H, a straight or branched carbonchain, preferably containing 1 to 6, more preferably 1 to 3, carbons,halide, alkoxyalkyl, or haloalkyl; and

R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹ and R¹² are each independentlyselected from among any of (i), (ii), (iii), (iv) or (v):

(i) R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹ and R¹² are each independentlyselected from among H, NHOH, NH₂, NO₂, N₃, halide, pseudohalide, alkyl,alkenyl, alkynyl, aryl, arylalkyl, heteroaryl, alkoxy, alkylamino,alkylthio, alkoxyalkyl, alkylsulfinyl, alkylsulfonyl, aryloxy,arylamino, arylthio, arylsulfinyl, arylsulfonyl, haloalkyl, haloaryl,alkoxycarbonyl, alkylcarbonyl, aminocarbonyl, arylcarbonyl, formyl,amido, ureido, or at least two of R³, R⁴, R⁵, R⁶ and R⁷ or R⁸, R⁹, R¹⁰,R¹¹ and R¹² form alkylenedioxy where the alkyl, alkenyl, alkynylportions are straight or branched chains of from about 1 up to about 10carbons, preferably, 1 to about 5 or 6 carbons and in which the arylportions contain from 3 up to about 10 carbons, preferably 6 carbons; or

(ii) R⁴ and R⁷ and/or R¹⁰ and R¹¹ together are substituted orunsubstituted 1,3-butadienyl, 4-dimethylamino-1,3-butadienyl,1-chloro-1,3-butadienyl, 1-aza-1,3-butadienyl or 2-aza-1,3-butadienylgroups; and the others of R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹ and R¹²are as defined in (i) above; or alternatively,

(iii) R⁷ and R³ and/or R¹¹ and R¹² together are substituted orunsubstituted 1,3-butadienyl, 4-dimethylamino-1,3-butadienyl,1-chloro-1,3-butadienyl, 1-aza-1,3-butadienyl or 2-aza-1,3-butadienylgroups; and the others of R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹ and R¹²are as defined in (i) above; or

(iv) R³, R⁵, and R⁷ and R¹⁰, R¹¹ and R⁸ are H or are as defined in (i);and R⁴ and R⁶ and/or R⁹ and R¹² are each independently selected fromalkyl, alkoxy, halide, aminoalkyl, dialkylaminoalkyl, in which the alkyland alkoxy groups contain from 1 to 10, preferably 1 to 6 carbons, andare straight or branched chains, and the others of R³, R⁴, R⁵, R⁶, R⁷,R⁸, R⁹, R¹⁰, R¹¹ and R¹² are as defined in (i) above; or

(v) any two of R³, R⁴, R⁵, R⁶, and R⁷, and/or any two of R⁸, R⁹, R¹⁰,R¹¹ and R¹², which are each selected as in (i) form fused carbocyclic orheterocyclic rings.

In particularly preferred embodiments described herein, the compoundshave formula III, in which R¹ and R² are independently selected fromamong lower alkyl, lower alkenyl, lower alkynyl, lower haloalkyl,halide, pseudohalide or H, with the proviso that R² is not halide orpseudohalide.

In the preferred compounds, X and Y are independently selected fromamong (CH₂)_(n) where n is 0 or 1, and O; and

R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹ and R¹² are independently selectedfrom among H, NH₂, halide, pseudohalide, lower alkyl, lower alkenyl andlower haloalkyl and at least three of R³, R⁴, R⁵, R⁶ and R⁷ are hydrogenand at least three of R⁸, R⁹, R¹⁰, R¹¹ and R¹² are hydrogen.

In more preferred compounds, R² is selected from among lower alkyl,lower alkenyl, lower alkynyl, lower haloalkyl; and R¹ is lower alkyl orhalide, particularly bromide or chloride.

In the presently preferred compounds, the compounds have formula III inwhich X and Y are independently O or S, preferably O, and o and p areboth either 0 or both 1. Compounds in which o and p are 1 arephosphoramidates, and compounds in which o and p are 0 are phosphinicamides.

1. Phosphoramidates for Use in Treating Endothelin-mediated Diseases

Compounds and methods for treating endothelin-mediated diseases usingthe compounds of formula III are provided. In particular, the compoundsof formula III in which X and Y are O have formula IV:

in which R¹ and R² are each independently selected from H, NHOH, NH₂,NO₂, N₃, halide, pseudohalide, alkyl, alkenyl, alkynyl, aryl, arylalkyl,heteroaryl, alkoxy, alkylamino, alkylthio, alkylsulfinyl, alkylsulfonyl,aryloxy, arylamino, arylthio, arylsulfinyl, arylsulfonyl, haloalkyl,haloaryl, alkoxycarbonyl, alkylcarbonyl, aminocarbonyl, arylcarbonyl,formyl, amido, ureido, in which the alkyl, alkenyl, alkynyl portions arestraight or branched chains of from about 1 up to about 10 carbons,preferably, 1 to about 5 or 6 carbons and in which the aryl portionscontain from 3 up to about 10 carbons, preferably 6 carbons; and

R³, R⁴, R⁵, R⁶, R⁷, R⁸ ₇ R⁹, R¹⁰, R¹¹ and R¹² preferably are eachindependently selected from among H, NHOH, NH₂, NO₂, N₃, halide,pseudohalide, alkyl, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl,alkoxy, alkylamino, alkylthio, alkoxyalkyl, alkylsulfinyl,alkylsulfonyl, aryloxy, arylamino, arylthio, arylsulfinyl, arylsulfonyl,haloalkyl, haloaryl, alkoxycarbonyl, alkylcarbonyl, aminocarbonyl,arylcarbonyl, formyl, amido, ureido, or at least two of R³, R⁴, R⁵, R⁶,R⁷, R⁸, R⁹, R¹⁰, R¹¹ and R¹² form alkylenedioxy, in which the alkyl,alkenyl, alkynyl portions are straight or branched chains of from about1 up to about 10 carbons, preferably, 1 to about 5 or 6 carbons and inwhich the aryl portions contain from 3 up to about 10 carbons,preferably 6 carbons are provided.

In more preferred of these embodiments, R¹ is H, halide, pseudohalide,lower alkyl, lower alkenyl or lower haloalkyl, and R² is H, lower alkyl,lower alkenyl and lower haloalkyl; and

R³, R⁴, R⁵, R⁵, R⁷, R⁸, R⁹, R¹⁰, R¹¹ and R¹² are independently selectedfrom among H, NH₂, halide, pseudohalide, lower alkyl, lower alkenyl andlower haloalkyl, and preferably, at least three of R³, R⁴, R⁵, R⁶ and R⁷are hydrogen and at least three of R⁸, R⁹, R¹⁰, R¹¹ and R¹² arehydrogen.

In the most preferred of these embodiments in which the compounds haveformula IV, R¹ is halide, pseudohalide or lower alkyl, preferably loweralkyl or Br or Cl; R² is lower alkyl, preferably methyl or ethyl; and

R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹ and R¹² are independently selectedfrom among H, NH₂, halide, pseudohalide, lower alkyl, lower alkenyl andlower haloalkyl, preferably lower alkyl or hydrogen, and most preferablyhydrogen.

2. Phosphinic Amides for Use in Treating Endothelin-mediated Diseases

Compounds and methods for treating endothelin-mediated diseases usingthe compounds of formula I are provided. In particular, compounds inwhich Ar¹ is 5-isoxazole or 3-isoxazole and A and B are each phenyl areprovided. In particular, compounds having formula V:

in which R¹ and R² are each independently selected from H, NHOH, NH₂,NO₂, N₃, halide, pseudohalide, alkyl, alkenyl, alkynyl, aryl, arylalkyl,heteroaryl, alkoxy, alkylamino, alkylthio, alkylsulfinyl, alkylsulfonyl,aryloxy, arylamino, arylthio, arylsulfinyl, arylsulfonyl, haloalkyl,haloaryl, alkoxycarbonyl, alkylcarbonyl, aminocarbonyl, arylcarbonyl,formyl, amido, ureido, or at least two of R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹,R¹⁰, R¹¹ and R¹² form alkylenedioxy, in which the alkyl, alkenyl,alkynyl portions are straight or branched chains of from about 1 up toabout 10 carbons, preferably 1 to about 5 or 6 carbons and in which thearyl portions contain from 3 up to about 10 carbons, preferably 6carbons; and

R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹ and R¹² are each independentlyselected from among H, NHOH, NH₂, NO₂, N₃, halide, pseudohalide, alkyl,alkenyl, alkynyl, aryl, arylalkyl, heteroaryl, alkoxy, alkylamino,alkylthio, alkoxyalkyl, alkylsulfinyl, alkylsulfonyl, aryloxy,arylamino, arylthio, arylsulfinyl, arylsulfonyl, haloalkyl, haloaryl,alkoxycarbonyl, alkylcarbonyl, aminocarbonyl, arylcarbonyl, formyl,amido, ureido, in which the alkyl, alkenyl, alkynyl portions arestraight or branched chains of from about 1 up to about 10 carbons,preferably, 1 to about 5 or 6 carbons and in which the aryl portionscontain from 3 up to about 10 carbons, preferably 6 carbons areprovided.

In the more preferred of the embodiments in which the compounds haveformula V, R¹ is halide, pseudohalide or lower alkyl, preferably loweralkyl or Br or Cl; R² is lower alkyl, preferably methyl or ethyl; and

R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹ and R¹² are independently selectedfrom among H, NH₂, halide, pseudohalide, lower alkyl, lower alkenyl andlower haloalkyl, preferably lower alkyl or hydrogen, and most preferablyhydrogen.

In more preferred of these embodiments, R¹ is halide, pseudohalide orlower alkyl, preferably lower alkyl or bromine and R² is lower alkyl,preferably methyl or ethyl; and

R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹ and R¹² are each independentlyselected from among H, NH₂, halide, pseudohalide, lower alkyl, loweralkenyl and lower haloalkyl, preferably lower alkyl or hydrogen, andmost preferably hydrogen.

B. Activities of the Compounds

Exemplary compounds were synthesized and tested using the exemplifiedassays (see, EXAMPLES) and selected results are set forth in Table 1:

TABLE 1 COMPOUND ET_(A) (μM)*^(†) ET_(B) (μM)*^(†)N-(3,4-dimethyl-5-isoxazolyl)- 2.5 >100 diphenylphosphoramidateN-(3,4-dimethyl-5-isoxozolyl)- 7.1 >100 diphenylphosphinic amideN-(4-bromo-3-methyl-5-isoxa- 0.58 77 zolyl)diphenylphosphoramidateN-(4-bromo-3-methyl-5-isoxa- 0.84 85 zolyl)diphenylphosphinic amideN-(4-bromo-3-methyl-5-isoxa- 20 >100 zolyl)diethylphosphoramidate*results generally from 1, 2, or 3 experiments with the same preparation^(\)assay performed with incubation at 4° C. As described in theExamples, incubation at the higher temperature reduces the activity by afactor of 2- to about 10-compared to the activity at 4° C. —data notavailable or measured as % inhibition @ 100 μM %= % inhibition @ 100 μM

C. Preparation of the Compounds

Preparation of exemplary compounds is described in detail in theexamples. The compounds provided herein may be synthesized according toa method discussed in general below and set forth in the Examples byselecting appropriate starting materials.

In general, the syntheses involve the condensation of a chlorophosphateor phosphinic chloride with an aminoisoxazole in dry pyridine or intetrahydrofuran (THF) and sodium hydride. The chlorophosphates orphosphinic chlorides and aminoisoxazoles either can be obtainedcommercially or synthesized according to methods available to those ofskill in this art.

In particular, the compounds may be prepared by reacting an appropriatephosphinic chloride or chlorophosphate with 5-aminoisoxazolessubstituted at the 3 and 4 positions, such as4-bromo-3-methyl-5-aminoisoxazole, in tetrahydrofuran (THF) solutioncontaining a base, such as sodium hydride. Following the reaction, themixture is diluted with ethyl acetate and washed with HCl. The organiclayer is dried over anhydrous magnesium sulfate, filtered andconcentrated. The residue is purified by flash chromatography usingethyl acetate/hexanes followed by recrystallization using chloroform andhexanes.

The N-(4-haloisoxazolyl)phosphoramidates andN-(4-haloisoxazolyl)-phosphinic amides can be prepared by condensationof an amino-4-haloisoxazole with a chlorophosphate or phosphinicchloride, respectively, in THF with sodium hydride as a base. Forexample, N-(4-bromo-3-methyl-5-isoxazolyl)diphenylphosphoramidate wasprepared from 4-bromo-3-methyl-5-aminoisoxazole and diphenylchlorophosphate in THF and sodium hydride.N-(4-bromo-3-methyl-5-isoxazolyl)diethylphosphor-amidate was preparedfrom 4-bromo-3-methyl-5-aminoisoxazole and diethyl chlorophosphate.

Alternatively, the phosphoramidates and/or phosphinic amides can beprepared from the corresponding phosphinic chloride or chlorophosphateand the appropriately substituted aminoisoxazole in pyridine. Followingthe reaction, the mixture is diluted with ethyl acetate, washed with HCland the organic layer dried, filtered and concentrated. The residue ispurified by flash chromatography using methanol/chloroform, followed byrecrystallization from ethyl acetate or chloroform and hexanes.

The N-(alkylisoxazolyl)phosphoramidates andN-(alkylisoxazolyl)phosphinic amides can be prepared by condensing analkylaminoisoxazole with a chlorophosphate or phosphinic chloride,respectively, in dry pyridine. For example, theN-(3,4-dimethyl-5-isoxazolyl)phosphoramidates andN-(3,4-dimethyl-5-isoxazolyl)phosphinic amides can be prepared from3,4-dimethyl-5-aminoisoxazole and the appropriately substitutedchlorophosphate or phosphinic chloride in pyridine.

Prodrugs and other derivatives of the compounds suitable foradministration to humans may also be designed and prepared by methodsknown to those of skill in the art (see, e.g., Nogrady (1985) MedicinalChemistry A Biochemical Approach, Oxford University Press, New York,pages 388-392).

Compounds listed and described have been synthesized and tested foractivity in in vitro assays and, in some cases, in in vivo animalmodels. Nuclear magnetic resonance spectroscopic (NMR), massspectrometric, infrared spectroscopic and high performance liquidchromatographic analyses indicated that the synthesized compounds havestructures consistent with those expected for such compounds and aregenerally at least about 98% pure. All of the compounds exemplified ordescribed herein exhibited activity as endothelin antagonists.

D. Evaluation of the Bioactivity of the Compounds

Standard physiological, pharmacological and biochemical procedures areavailable for testing the compounds to identify those that possess anybiological activities of an endothelin peptide or the ability tointerfere with or inhibit endothelin peptides. Compounds that exhibit invitro activities, such as the ability to bind to endothelin receptors orto compete with one or more of the endothelin peptides for binding toendothelin receptors can be used in the methods for isolation ofendothelin receptors and the methods for distinguishing thespecificities of endothelin receptors, and are candidates for use in themethods of treating endothelin-mediated disorders.

Thus, compounds of formulae I-V that are endothelin antagonists oragonists may be identified using such screening assays.

1. Identifying Compounds that Modulate the Activity of an EndothelinPeptide

The compounds are tested for the ability to modulate the activity ofendothelin-1. Numerous assays are known to those of skill in the art forevaluating the ability of compounds to modulate the activity ofendothelin (see, e.g., U.S. Pat. No. 5,114,918 to Ishikawa et al.; EP A10 436 189 to BANYU PHARMACEUTICAL CO., LTD. (Oct. 7, 1991); Borges etal. (1989) Eur. J. Pharm. 165: 223-230; Filep et al. (1991) Biochem.Biophys. Res. Commun. 177: 171-176). In vitro studies may becorroborated with in vivo studies (see, e.g., U.S. Pat. No. 5,114,918 toIshikawa et al.; EP A1 0 436 189 to BANYU PHARMACEUTICAL CO., LTD. (Oct.7, 1991)) and pharmaceutical activity thereby evaluated. Such assays aredescribed in the Examples herein and include the ability to compete forbinding to ET_(A) and ET_(B) receptors present on membranes isolatedfrom cell lines that have been genetically engineered to express eitherET_(A) or ET_(B) receptors on their cell surfaces.

The properties of a potential antagonist may be assessed as a functionof its ability to inhibit an endothelin induced activity in vitro usinga particular tissue, such as rat portal vein and aorta as well as ratuterus, trachea and vas deferens (see e.g., Borges, R., Von Grafenstein,H. and Knight, D. E., Tissue selectivity of endothelin, Eur. J.Pharmacol 165:223-230, (1989)). The ability to act as an endothelinantagonist in vivo can be tested in hypertensive rats, ddy mice or otherrecognized animal models (see, Kaltenbronn et al. (1990) J. Med. Chem.33:838-845, see, also, U.S. Pat. No. 5,114,918 to Ishikawa et al.; andEP A1 0 436 189 to BANYU PHARMACEUTICAL CO., LTD (Oct. 7, 1991); see,also Bolger et al. (1983) J. Pharmacol. Exp. Ther. 225291-309). Usingthe results of such animal studies, pharmaceutical effectiveness may beevaluated and pharmaceutically effective dosages determined. A potentialagonist may also be evaluated using in vitro and in vivo assays known tothose of skill in the art.

Endothelin activity can be identified by the ability of a test compoundto stimulate constriction of isolated rat thoracic aorta (Borges et al.(1989) “Tissue selectivity of endothelin” Eur. J. Pharmacol. 165:223-230). To perform the assay, the endothelium is abraded and ringsegments mounted under tension in a tissue bath and treated withendothelin in the presence of the test compound. Changes in endothelininduced tension are recorded. Dose response curves may be generated andused to provide information regarding the relative inhibitory potency ofthe test compound. Other tissues, including heart, skeletal muscle,kidney, uterus, trachea and vas deferens, may be used for evaluating theeffects of a particular test compound on tissue contraction.

Endothelin isotype specific antagonists may be identified by the abilityof a test compound to interfere with endothelin binding to differenttissues or cells expressing different endothelin-receptor subtypes, orto interfere with the biological effects of endothelin or an endothelinisotype (Takayanagi et al. (1991) Reg. Pep. 32: 23-37, Panek et al.(1992) Biochem. Biophys. Res. Commun. 183: 566-571). For example, ET_(B)receptors are expressed in vascular endothelial cells, possiblymediating the release of prostacyclin and endothelium-derived relaxingfactor (De Nucci et al. (1988) Proc. Natl. Acad. Sci. USA 85:9797).ET_(A) receptors are not detected in cultured endothelial cells, whichexpress ET_(B) receptors.

The binding of compounds or inhibition of binding of endothelin toET_(B) receptors can be assessed by measuring the inhibition ofendothelin-1-mediated release of prostacyclin, as measured by its majorstable metabolite, 6-keto PGF_(1α), from cultured bovine aorticendothelial cells (see, e.g., Filep et al. (1991) Biochem. and BiophysRes. Commun. 177: 171-176). Thus, the relative affinity of the compoundsfor different endothelin receptors may be evaluated by determining theinhibitory dose response curves using tissues that differ in receptorsubtype.

Using such assays, the relative affinities of the compounds for ET_(A)receptors and ET_(B) receptors have been and can be assessed. Those thatpossess the desired properties, such as specific inhibition of bindingof endothelin-1, are selected. The selected compounds that exhibitdesirable activities may be therapeutically useful and are tested forsuch uses using the above-described assays from which in vivoeffectiveness may be evaluated (see, e.g., U.S. Pat. No. 5,248,807; U.S.Pat. No. 5,240,910; U.S. Pat. No. 5,198,548; U.S. Pat. No. 5,187,195;U.S. Pat. No. 5,082,838; U.S. Pat. No. 5,230,999; published CanadianApplication Nos. 2,067,288 and 2071193; published Great BritainApplication No. 2,259,450; Published International PCT Application No.WO 93/08799; Benigi et al. (1993) Kidney International 44:440-444; andNirei et al. (1993) Life Sciences 52:1869-1874). Compounds that exhibitin vitro activities that correlate with in vivo effectiveness will thenbe formulated in suitable pharmaceutical compositions and used astherapeutics.

The compounds also may be used in methods for identifying and isolatingendothelin-specific receptors and aiding in the design of compounds thatare more potent endothelin antagonists or agonists or that are morespecific for a particular endothelin receptor.

2. Isolation of Endothelin Receptors

A method for identifying endothelin receptors is provided. In practicingthis method, one or more of the compounds is linked to a support andused in methods of affinity purification of receptors. By selectingcompounds with particular specificities, distinct subclasses of ETreceptors may be identified.

One or more of the compounds may be linked to an appropriate resin, suchas Affi-gel, covalently or by other linkage, by methods known to thoseof skill in the art for linking endothelin to such resins (see, Schvartzet al. (1990) Endocrinology 126: 3218-3222). The linked compounds can bethose that are specific for ET_(A) or ET_(B) receptors or other subclassof receptors.

The resin is pre-equilibrated with a suitable buffer generally at aphysiological pH (7 to 8). A composition containing solubilizedreceptors from a selected tissue are mixed with the resin to which thecompound is linked and the receptors are selectively eluted. Thereceptors can be identified by testing them for binding to an endothelinisopeptide or analog or by other methods by which proteins areidentified and characterized. Preparation of the receptors, the resinand the elution method may be performed by modification of standardprotocols known to those of skill in the art (see, e.g., Schvartz et al.(1990) Endocrinology 126: 3218-3222).

Other methods for distinguishing receptor type based on differentialaffinity to any of the compounds herein are provided. Any of the assaysdescribed herein for measuring the affinity of selected compounds forendothelin receptors may also be used to distinguish receptors subtypesbased on affinity for particular compounds provided herein. Inparticular, an unknown receptor may be identified as an ET_(A) or ET_(B)receptor by measuring the binding affinity of the unknown receptor for acompound provided herein that has a known affinity for one receptor overthe other. Such preferential interaction is useful for determining theparticular disease that may be treated with a compound prepared asdescribed herein. For example, compounds with high affinity for ET_(A)receptors and little or no affinity for ET_(B) receptors are candidatesfor use as hypertensive agents; whereas, compounds that preferentiallyinteract with ET_(B) receptors are candidates for use as anti-asthmaagents.

E. Formulation and Administration of the Compositions

Effective concentrations of one or more of the phosphoramidate orphosphinic amide compounds of formulae I-V or pharmaceuticallyacceptable salts, esters or other derivatives thereof are mixed with asuitable pharmaceutical carrier or vehicle.

In instances in which the compounds exhibit insufficient solubility,methods for solubilizing compounds may be used. Such methods are knownto those of skill in this art, and include, but are not limited to,using cosolvents, such as dimethylsulfoxide (DMSO), using surfactants,such as tween, or dissolution in aqueous sodium bicarbonate. Derivativesof the compounds, such as salts of the compounds or prodrugs of thecompounds may also be used in formulating effective pharmaceuticalcompositions.

The concentrations of the compounds are effective for delivery of anamount, upon administration, that ameliorates the symptoms of theendothelin-mediated disease. Typically, the compositions are formulatedfor single dosage administration.

Upon mixing or addition of the phosphoramidate or phosphinic amidecompound(s), the resulting mixture may be a solution, suspension,emulsion or the like. The form of the resulting mixture depends upon anumber of factors, including the intended mode of administration and thesolubility of the compound in the selected carrier or vehicle. Theeffective concentration is sufficient for ameliorating the symptoms ofthe disease, disorder or condition treated and may be empiricallydetermined.

Pharmaceutical carriers or vehicles suitable for administration of thecompounds provided herein include any such carriers known to thoseskilled in the art to be suitable for the particular mode ofadministration. In addition, the compounds may be formulated as the solepharmaceutically active ingredient in the composition or may be combinedwith other active ingredients.

The active compounds can be administered by any appropriate route, forexample, orally, parenterally, intravenously, intradermally,subcutaneously, or topically, in liquid, semi-liquid or solid form andare formulated in a manner suitable for each route of administration.Preferred modes of administration include oral and parenteral modes ofadministration.

The active compound is included in the pharmaceutically acceptablecarrier in an amount sufficient to exert a therapeutically useful effectin the absence of undesirable side effects on the patient treated. Thetherapeutically effective concentration may be determined empirically bytesting the compounds in known in vitro and in vivo systems (see, e.g.,U.S. Pat. No. 5,114,918 to Ishikawa et al.; EP A1 0 436 189 to BANYUPHARMACEUTICAL CO., LTD (Oct. 7, 1991); Borges et al. (1989) Eur. J.Pharm. 165: 223-230;: Filep et al. (1991) Biochem. Biophys. Res. Commun.177: 171-176) and then extrapolated therefrom for dosages for humans.

The concentration of active compound in the drug composition will dependon absorption, inactivation and excretion rates of the active compound,the dosage schedule, and amount administered as well as other factorsknown to those of skill in the art. For example, the amount that isdelivered is sufficient to treat the symptoms of hypertension.

Typically a therapeutically effective dosage should produce a serumconcentration of active ingredient of from about 0.1 ng/ml to about50-100 μg/ml. The pharmaceutical compositions typically should provide adosage of from about 0.01 mg to about 2000 mg of compound per kilogramof body weight per day. The active ingredient may be administered atonce, or may be divided into a number of smaller doses to beadministered at intervals of time. It is understood that the precisedosage and duration of treatment is a function of the disease beingtreated and may be determined empirically using known testing protocolsor by extrapolation from in vivo or in vitro test data. It is to benoted that concentrations and dosage values may also vary with theseverity of the condition to be alleviated. It is to be furtherunderstood that for any particular subject, specific dosage regimensshould be adjusted over time according to the individual need and theprofessional judgment of the person administering or supervising theadministration of the compositions, and that the concentration rangesset forth herein are exemplary only and are not intended to limit thescope or practice of the claimed compositions.

If oral administration is desired, the compound should be provided in acomposition that protects it from the acidic environment of the stomach.For example, the composition can be formulated in an enteric coatingthat maintains its integrity in the stomach and releases the activecompound in the intestine. The composition may also be formulated incombination with an antacid or other such ingredient.

Oral compositions will generally include an inert diluent or an ediblecarrier and may be compressed into tablets or enclosed in gelatincapsules. For the purpose of oral therapeutic administration, the activecompound or compounds can be incorporated with excipients and used inthe form of tablets, capsules or troches. Pharmaceutically compatiblebinding agents and adjuvant materials can be included as part of thecomposition.

The tablets, pills, capsules, troches and the like can contain any ofthe following ingredients, or compounds of a similar nature: a binder,such as microcrystalline cellulose, gum tragacanth and gelatin; anexcipient such as starch and lactose, a disintegrating agent such as,but not limited to, alginic acid and corn starch; a lubricant such as,but not limited to, magnesium stearate; a glidant, such as, but notlimited to, colloidal silicon dioxide; a sweetening agent such assucrose or saccharin; and a flavoring agent such as peppermint, methylsalicylate, and fruit flavoring.

When the dosage unit form is a capsule, it can contain, in addition tomaterial of the above type, a liquid carrier such as a fatty oil. Inaddition, dosage unit forms can contain various other materials whichmodify the physical form of the dosage unit, for example, coatings ofsugar and other enteric agents. The compounds can also be administeredas a component of an elixir, suspension, syrup, wafer, chewing gum orthe like. A syrup may contain, in addition to the active compounds,sucrose as a sweetening agent and certain preservatives, dyes andcolorings and flavors.

The active materials can also be mixed with other active materials whichdo not impair the desired action, or with materials that supplement thedesired action, such as antacids, H2 blockers, and diuretics. Forexample, if the compound is used for treating asthma or hypertension, itmay be used with other bronchodilators and antihypertensive agents,respectively.

Solutions or suspensions used for parenteral, intradermal, subcutaneous,or topical application can include any of the following components: asterile diluent, such as water for injection, saline solution, fixedoil, polyethylene glycol, glycerine, propylene glycol or other syntheticsolvent; antimicrobial agents, such as benzyl alcohol and methylparabens; antioxidants, such as ascorbic acid and sodium bisulfite;chelating agents, such as ethylenediaminetetraacetic acid (EDTA);buffers, such as acetates, citrates and phosphates; and agents for theadjustment of tonicity such as sodium chloride or dextrose. Parentalpreparations can be enclosed in ampules, disposable syringes or multipledose vials made of glass, plastic or other suitable material.

If administered intravenously, suitable carriers include physiologicalsaline or phosphate buffered saline (PBS), and solutions containingthickening and solubilizing agents, such as glucose, polyethyleneglycol, and polypropylene glycol and mixtures thereof. Liposomalsuspensions, including tissue-targeted liposomes, may also be suitableas pharmaceutically acceptable carriers. These may be prepared accordingto methods known to those skilled in the art. For example, liposomeformulations may be prepared as described in U.S. Pat. No. 4,522,811.

The active compounds may be prepared with carriers that protect thecompound against rapid elimination from the body, such as time releaseformulations or coatings. Such carriers include controlled releaseformulations, such as, but not limited to, implants andmicroencapsulated delivery systems, and biodegradable, biocompatiblepolymers, such as collagen, ethylene vinyl acetate, polyanhydrides,polyglycolic acid, polyorthoesters, polylactic acid and others. Methodsfor preparation of such formulations are known to those skilled in theart.

The compounds may be formulated for local or topical application, suchas for topical application to the skin and mucous membranes, such as inthe eye, in the form of gels, creams, and lotions and for application tothe eye or for intracisternal or intraspinal application. Suchsolutions, particularly those intended for ophthalmic use, may beformulated as 0.01%-10% isotonic solutions, pH about 5-7, withappropriate salts. The compounds may be formulated as aerosols fortopical application, such as by inhalation (see, e.g., U.S. Pat. Nos.4,044,126, 4,414,209, and 4,364,923, which describe aerosols fordelivery of a steroid useful for treatment inflammatory diseases,particularly asthma).

Finally, the compounds may be packaged as articles of manufacturecontaining packaging material, a compound provided herein, which iseffective for antagonizing the effects of endothelin, ameliorating thesymptoms of an endothelin-mediated disorder, or inhibiting binding of anendothelin peptide to an ET receptor with an IC₅₀ of less than about 50μM, preferably about 20 μM or less, more preferably less than 10 μM,within the packaging material, and a label that indicates that thecompound or salt thereof is used for antagonizing the effects ofendothelin, treating endothelin-mediated disorders or inhibiting thebinding of an endothelin peptide to an ET receptor.

The following examples are included for illustrative purposes only andare not intended to limit the scope of the invention.

EXAMPLE 1 N-(3,4-dimethyl-5-isoxazolyl)diphenylphosphoramidate

3,4-Dimethyl-5-aminoisoxazole (0.34 g, 3.0 mmol) and diphenylchlorophosphate (0.62 ml 3.3 mmol) were stirred in pyridine (7 ml) atambient temperature for 1 h, then diluted with ethyl acetate (100 ml)and washed with 2% HCl (3×100 ml). The organic layer was dried (MgSO₄),filtered and concentrated to collect 0.73 g of a light orange oil. Flashchromatography (2% methanol/chloroform) followed by recrystallizationfrom chloroform and hexanes provided 0.31 g (30%) of white crystals,m.p. 97-99° C.

EXAMPLE 2 N-(3,4-dimethyl-5-isoxazolyl)diphenylphosphinic Amide

N-(3,4-dimethyl-5-isoxazolyl)diphenyl phosphinic amide was prepared bythe method of Example 1 with 3,4-dimethyl-5-amino isoxazole (0.22 g, 2.0mmol), diphenyl phosphinic chloride (0.6 ml, 3.0 mmol) and pyridine (5ml). Flash chromatography (5% methanol/chloroform) followed byrecrystallization from ethyl acetate and hexanes provided 0.38 g (61%)of white crystals, m.p. 167-170° C.

EXAMPLE 3 N-(4-bromo-3-methyl-5-isoxazolyl)diphenylphosphoramidate

NaH (60% oil dispersion, 84 mg, 2.1 mmol) was added to4-bromo-3-methyl-5-amino isoxazole (0.18 g, 1.0 mmol) in THF (5 ml) atambient temperature. After 10 min. of stirring diphenyl chlorophosphate(0.23 ml, 1.1 mmol) was added. The reaction solution was stirred 1 hr atambient temperature, then diluted with ethyl acetate (100 mls) andwashed with 2% HCl (2×100 ml). The organic was dried (MgSO₄), filteredand concentrated. Flash chromatography (50% ethyl acetate/hexane)followed by recrystallization from chloroform and hexanes provided 0.26g (64%) of tan crystals, m.p. 116.5-118.5° C.

EXAMPLE 4 N-(4-bromo-3-methyl-5-isoxazolyl)diphenylphosphinic Amide

N-(4-bromo-3-methyl-5-isoxazolyl)diphenyl phosphinic amide was preparedby the method of Example 3 with 4-bromo-3-methyl-5-amino isoxazole (0.18g, 1.0 mmol), THF (5 ml), NaH (60% oil dispersion, 88 mg, 2.2 mmol), anddiphenyl phosphinic chloride (0.23 ml, 1.2 mmol). Recrystallizationthree times from chloroform and hexanes provided 0.17 g (45%) of faintpink crystals, m.p. 178.5-181° C.

EXAMPLE 5 N-(4-bromo-3-methyl-5-isoxazolyl)diethylphosphoramidate

N-(4-bromo-3-methyl-5-isoxazolyl)diethyl phosphoramidate was prepared bythe method of Example 3 with 4-bromo-3-methyl-5-amino isoxazole (0.27 g,1.5 mmol), THF (8 ml), NaH (60% oil dispersion, 0.15 g, 3.8 mmol) anddiethyl chlorophosphate (0.25 ml, 1.7 mmol). Recrystallization two timesfrom chloroform and hexanes provided 0.24 g (51%) of light yellowcrystals, m.p. 78-80° C.

EXAMPLE 6 Assays for Identifying Compounds that Exhibit EndothelinAntagonistic and/or Agonist Activity

Compounds that are potential endothelin antagonists are identified bytesting their ability to compete with ¹²⁵I-labeled ET-1 for binding tohuman ET_(A) receptors or ET_(B) receptors present on isolated cellmembranes. The effectiveness of the test compound as an antagonist oragonist of the biological tissue response of endothelin can also beassessed by measuring the effect on endothelin induced contraction ofisolated rat thoracic aortic rings. The ability of the compounds to actas antagonists or agonists for ET_(B) receptors can be assessed bytesting the ability of the compounds to inhibit endothelin-1 inducedprostacyclin release from cultured bovine aortic endothelial cells.

A. Endothelin Binding Inhibition—Binding Test #1: Inhibition of Bindingto ET_(A) Receptors

TE 671 cells (ATCC Accession No. HTB 139) express ET_(A) receptors.These cells were grown to confluence in T-175 flasks. Cells frommultiple flasks were collected by scraping, pooled and centrifuged for10 min at 190×g. The cells were resuspended in phosphate buffered saline(PBS) containing 10 mM EDTA using a Tenbroeck homogenizer. Thesuspension was centrifuged at 4° C. at 57,800×g for 15 min, the pelletwas resuspended in 5 ml of buffer A (5 mM HEPES buffer, pH 7.4containing aprotinin (100 KIU/ml)) and then frozen and thawed once. 5 mlof Buffer B (5 mM HEPES Buffer, pH 7.4 containing 10 mM MnCl₂ and 0.001%deoxyribonuclease Type 1) was added, the suspension mixed by inversionand then incubated at 37° C. for 30 minutes. The mixture was centrifugedat 57,800×g as described above, the pellet washed twice with buffer Aand then resuspended in buffer C (30 mM HEPES buffer, pH 7.4 containingaprotinin (100 KIU/ml) to give a final protein concentration of 2 mg/mland stored at −70° C. until use.

The membrane suspension was diluted with binding buffer (30 mM HEPESbuffer, pH 7.4 containing 150 mM NaCl, 5 mM MgCl₂, 0.5% Bacitracin) to aconcentration of 8 μg/50 μl. ¹²⁵I-endothelin-1 (3,000 cpm, 50 mL) wasadded to 50 μL of either: (A) endothelin-1 (for non specific binding) togive a final concentration 80 nM); (B) binding buffer for totalbinding); or (C) a test compound (final concentration 1 nM to 100 μM).The membrane suspension (50 μL), containing up to 8 μg of membraneprotein, was added to each of (A), (B), or (C). Mixtures were shaken,and incubated at 4° C. for 16-18 hours, and then centrifuged at 4° C.for 25 min at 2,500×g. Alternatively, the incubation can be conducted atother temperatures, such as 24° C. When incubated at 24° C., the IC₅₀concentrations are 2- to 10-fold higher than when the incubation isconducted at 4° C.

The supernatant, containing unbound radioactivity, was decanted and thepellet counted on a Genesys multiwell gamma counter. The degree ofinhibition of binding (D) was calculated according to the followingequation:${\% \quad D} = {100 - {\frac{(C) - (A)}{(B) - (A)} \times 100}}$

Each test was generally performed in triplicate.

B. Endothelin Binding Inhibition—Binding Test #2: Inhibition of Bindingto ET_(B) Receptors

COS7 cells were transfected with DNA encoding the ET_(B) receptor, Theresulting cells, which express the human ET_(B) receptor, were grown toconfluence in T-150 flasks. Membrane was prepared as described above.The binding assay was performed as described above using the membranepreparation diluted with binding buffer to a concentration of 1 μg/50μl.

Briefly, the COS7 cells, described above, that had been transfected withDNA encoding the ET_(B) receptor and express the human ET_(B) receptoron their surfaces were grown to confluence in T-175 flasks. Cells frommultiple flasks were collected by scraping, pooled and centrifuged for10 min at 190×g. The cells were resuspended in phosphate buffered saline(PBS) containing 10 mM EDTA using a Tenbroeck homogenizer. Thesuspension was centrifuged at 4° C. 57,800×g for 15 min, the pellet wasresuspended in 5 ml of buffer A (5 mM HEPES buffer, pH 7.4 containingaprotinin (100 KIU/ml)) and then frozen and thawed once. Five ml ofBuffer B (5 mM HEPES Buffer, pH 7.4 containing 10 mM MnCl₂ and 0.001%deoxyribonuclease Type 1) was added, the suspension mixed by inversionand then incubated at 37° C. for 30 minutes. The mixture was centrifugedat 57,800×g as described above, the pellet washed twice with buffer Aand then resuspended in buffer C (30 mM HEPES buffer, pH 7.4 containingaprotinin (100 KIU/ml) to give a final protein concentration of 2 mg/ml.

The binding assay was performed as described above using the membranepreparation diluted to give 1 μg/50 μl of binding buffer.

C. Test for Activity Against Endothelin-induced Contraction of IsolatedRat Thoracic Aortic Rings

The effectiveness of the test compound as an antagonist or agonist ofthe biological tissue response of endothelin also is assessed bymeasuring the effect on endothelin induced contraction of isolated ratthoracic aortic rings (see, e.g., Borges et al. (1989) Eur. J.Pharmacol. 165:223-230) or by measuring the ability to contract thetissue when added alone.

Compounds to be tested are prepared as 100 μM stocks. If necessary toeffect dissolution, the compounds are first dissolved in a minimumamount of DMSO and diluted with 150 mM NaCl. Because DMSO can causerelaxation of the aortic ring, control solutions containing varyingconcentrations of DMSO were tested.

The thoracic portion of the adult rat aorta is excised, the endotheliumabraded by gentle rubbing and then cut into 3 mm ring segments. Segmentsare suspended under a 2 g preload in a 10 ml organ bath filled withKrebs'-Henseleit solution saturated with a gas mixture of 95% O₂ and 5%CO₂ (118 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO₄, 1.2 mM KH₂PO₄, 25 mM NaHCO₃,2.5 mM CaCl₂, 10 mM D-glucose).

There is a correlation between activity as an antagonist ofendothelin-induced thoracic aortic ring contraction and activity as aninhibitor of binding of endothelin to endothelin receptors. The pA₂ is alinear function of the log of the IC₅₀.

D. Assay for Identifying Compounds that have Agonist and/or AntagonisticActivity Against ET_(B) Receptors

1. Stimulation of Prostacyclin Release

Since endothelin-1 stimulates the release of prostacyclin from culturedbovine aortic endothelial cells, the compounds that have agonist orantagonist activity are identified by their ability to inhibitendothelin-1 induced prostacyclin release from such endothelial cells bymeasuring 6-keto PGF_(1α) substantially as described by (Filep et al.(1991) Biochem. Biophys. Res. Commun. 177 171-176. Bovine aortic cellsare obtained from collagenase-treated bovine aorta, seeded into cultureplates, grown in Medium 199 supplemented with heat inactivated 15% fetalcalf serum, and L-glutamine (2 mM), penicillin, streptomycin andfungizone, and subcultured at least four times. The cells are thenseeded in six-well plates in the same medium. Eight hours before theassay, after the cells reach confluence, the medium is replaced. Thecells are then incubated with a) medium alone, b) medium containingendothelin-1 (10 nM), c) test compound alone, and d) testcompound+endothelin-1 (10 nM).

After a 15 min incubation, the medium is removed from each well and theconcentrations of 6-keto PGF_(1α) are measured by a direct immunoassay.Prostacyclin production is calculated as the difference between theamount of 6-keto PGF_(1α) released by the cells challenged with theendothelin-1 minus the amount released by identically treatedunchallenged cells. Compounds that stimulate 6-keto PGF_(1α) releasepossess agonist activity and those which inhibit endothelin-1 6-ketoPGF_(1α) release possess antagonist activity.

2. Inhibition of Sarafotoxin 6c Induced Contraction

Sarafotoxin 6c is a specific ET_(B) antagonist that contracts rat fundalstomach strips. The effectiveness of tests compounds to inhibit thissarafotoxin 6c-induced contraction of rat fundal stomach strips is usedas a measure ET_(B) antagonist activity. Two isolated rat fundal stomachstrips are suspended under a 1 g load in a 10 ml organ bath filled withKrebs'-Henseleit solution containing 10 μMcyclo(D-Asp-Pro-D-Val-Leu-D-Trp) (BQ-123; see, U.S. Pat. No. 5,114,918to Ishikawa et al.), 5 μM indomethacin, and saturated with a gas mixtureof 95% O₂/5% CO₂. Changes in tension are measured isometrically andrecorded using a Grass Polygraph coupled to a force transducer.Sarafotoxin 6c is added cumulatively to one strip while the second stripis preincubated for 15 min with a test compound prior to addition ofcumulative doses of sarafotoxin 6c. The effects of the test compounds onthe concentration-response curve for sarafotoxin 6c are examined.

E. Deoxycorticosterone Acetate (DOCA)-salt Hypertensive Rat Model forAssessing In Vivo Activity of Compounds

Another model useful for assessing the in vivo activity of compounds isthe deoxycorticosterone acetate (DOCA)-salt hypertensive rat model. Toperform these tests, silastic MDX4-4210 elastomer implants containing 47mg (DOCA) are prepared according to the method of Ornmsbee et al.((1973) the J. Pharm. Sci. 62:255-257). Briefly, DOCA is incorporatedinto silicon rubber implants for sustained release. To prepare theimplants the DOCA is incorporated into unpolymerized silicone rubber,catalyst is added and the mixture is cast in a hemicylindrical shape.

Sprague Dawley rats (7-8 weeks old) are unilaterally nephrectomizedunder ketamine anesthesia and a DOCA-implant is placed on the leftlateral dorsal abdomen of the animal. The rats are allowed to recoverfor three weeks. During recovery they are permitted free access tonormal rat chow and 0.9% NaCl drinking solution in place of drinkingwater. The rats develop hypertension within 3 weeks.

All animals are used in the tests between 21 and 30 days post surgery.The mean arterial blood pressure in these animals ranges from 165-200 mmHg.

On the day of experimentation, catheters are inserted under brevitalanesthesia into the right femoral artery for measurement of bloodpressure, and into the right femoral vein for administration of aselected compound. The animals are placed in a restrainer and allowed torecover for a minimum of 60 min or until a steady mean arterial bloodpressure is recorded. At that time, the selected compound or controlvehicle is administered either intravenously, as a 60 minute infusion,or orally by oral gavage. Blood pressure is recorded continuously for afurther 10 hrs.

F. Effect of Intravenous Administration on ET-1-induced PressorResponses in Conscious, Autonomically Blocked Rats; a Model forAssessing In Vivo Activity of Selected Compounds

Male Sprague Dawley rats (250-450 g) are anesthetized (Brevital 50mg/kg, IP) and cannulae were placed in the femoral artery to measuremean arterial pressure (MAP) and in the femoral vein for intravenousdrug administration. Animals are placed in a restrainer and allowed toregain consciousness. Thirty minutes later autonomic blockade isadministered (atropine methyl nitrate, 3 mg/kg, IV, followed bypropranalol, 2 mg/kg, IV). An hour later animals receive a bolusinjection of vehicle (0.5 ml) followed thirty minutes later byintravenous bolus administration of ET-1 (Control, 1 μg/kg). Followingrecovery from this challenge, test-compounds are administered byintravenous bolus administration (0.5 ml) and then re-challenged withET-1 thirty minutes later. Results are expressed as the percentinhibition of the ET-1-induced pressor response after administration ofthe test compound compared to the pressor response induced by thecontrol ET-1 challenge. In some cases a third ET-1 challenge isadministered ninety minutes after administration of the test compound.

G. Results

The IC₅₀ for each of the compounds of the preceding Examples for ET_(A)and ET_(B) receptors has been measured. Most of the compounds have anIC₅₀ of less than 10 μM (measured at 4° C.) for either or both of theET_(A) and ET_(B) receptors. The tested compounds have an IC₅₀ less thanabout 10-20 μM, some have an IC₅₀ less than about 5 μM and some of thecompounds have an IC₅₀ less than about 1 μM. A number of the compounds,such as N-(4-bromo-3-methyl-5-isoxazolyl)diphenylphosphoramidate andN-(4-bromo-3-methyl-5-isoxazolyl)diphenylphosphoramidate have an lC₅₀for ET_(A) receptors that is substantially less (10 to 100-fold or more)than for ET_(B) receptors, and, thus are selective for ET_(A) receptors.

Since modifications will be apparent to those of skill in this art, itis intended that this invention be limited only by the scope of theappended claims.

We claim:
 1. A compound of formula I:

or a pharmaceutically acceptable salt or ester thereof, wherein: Ar¹ isan aryl group containing one ring up to three fused rings and from 3 upto about 21 members in the ring(s); and A and B are independentlyselected from among pseudohalide, alkenyl, alkynyl, aryl, arylalkyl,heteroaryl, thioalkoxy, alkylamino, alkylthio, alkylsulfinyl,alkylsulfonyl, aryloxy, arylamino, arylthio, arylsulfinyl, arylsulfonyl,heteroaryloxy, heteroarylamino, heteroarylthio, haloalkyl, haloaryl,alkoxycarbonyl, alkylcarbonyl, aminocarbonyl, arylcarbonyl, formyl,amido and ureido, in which the alkyl, alkenyl and alkynyl portionscontain from 1 up to about 14 carbon atoms, and are either straight orbranched chains or cyclic, and in which the aryl and heteroaryl portionscontain from 4 to 16 members in the ring.
 2. A compound of claim 1,wherein Ar¹ is selected from naphthyl, phenyl, and biphenyl.
 3. Acompound of claim 1, wherein: Ar¹ is a 5 to 6 membered aryl group.
 4. Acompound of claim 1, wherein Ar¹ is unsubstituted or is substituted withone or more substituents selected from among NH₂, NO₂, halide,pseudohalide, alkyl, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl,alkoxy, alkylamino, alkylthio, alkylsulfinyl, alkylsulfonyl, aryloxy,arylamino, arylthio, arylsulfinyl, arylsulfonyl, haloalkyl, haloaryl,alkoxycarbonyl, alkylcarbonyl, aminocarbonyl, arylcarbonyl, formyl,substituted or unsubstituted amido and substituted or unsubstitutedureido, in which the alkyl, alkenyl and alkynyl portions contain from 1up to about 14 carbon atoms and are either straight or branched chainsor cyclic, and the aryl and heteroaryl portions contain from about 4 toabout 16 members in the ring.
 5. The compounds of claim 1, wherein Ar¹is selected from:

wherein: each group is unsubstituted or substituted with one or moresubstituents R; and each R, which may be the same or different, isindependently selected from NH₂, NO₂, halide, pseudohalide, alkyl,alkenyl, alkynyl, aryl, arylalkyl, heteroaryl, alkoxy, alkylamino,alkylthio, alkylsulfinyl, alkylsulfonyl, aryloxy, arylamino, arylthio,arylsulfinyl, arylsulfonyl, haloalkyl, haloaryl, alkoxycarbonyl,alkylcarbonyl, aminocarbonyl, arylcarbonyl, formyl, substituted orunsubstituted amido and substituted or unsubstituted ureido, in whichthe alkyl, alkenyl and alkynyl portions contain from 1 up to about 14carbon atoms and are either straight or branched chains or cyclic, andthe aryl and heteroaryl portions contain from about 4 to about 16members.
 6. A compound of claim 5, wherein each R is selected from amongH, halide, pseudohalide, alkyl, alkoxy, alkenyl or alkynyl.
 7. Acompound of formula I:

or a pharmaceutically acceptable salt or ester thereof, wherein: Ar¹ isan aryl group containing one ring up to three fused rings and from 3 upto about 21 members in the ring(s); and A has the formula Ar²—X_(p) andand B has the formula Ar³—Y_(o), wherein: o and p are independently 0 or1; Ar² and Ar³ are each independently selected from aryl and heteroarylgroups; X and Y are each independently selected from among O, N, NR ³, Sor (CH₂)_(n) in which n is 0 to 10; R¹³ is hydrogen or a groupcontaining up to about 30 carbon atoms selected from alkyl, alkenyl,alkynyl, aryl, alkylaryl, heterocyclyl, aralkyl, aralkoxy, cycloalkyl,cycloalkenyl, cycloalkynyl, C(O)R¹⁴ and S(O)_(n)R¹⁴ in which n is 0-2;R¹⁴ is hydrogen, alkyl, alkenyl, alkynyl, aryl, alkylaryl, heterocyclyl,aralkyl, aralkoxy, cycloalkyl, cycloalkenyl or cycloalkynyl; and R¹³ andR¹⁴ are unsubstituted or are substituted with one or more substituentseach selected independently from Z, which is hydrogen, halide,pseudohalide, alkyl, alkoxy, alkenyl, alkynyl, aryl, heterocyclyl,alkoxyalkyl, haloalkyl, aralkyl, aralkoxy, cycloalkyl, cycloalkenyl orcycloalkynyl.
 8. A compound of claim 7, wherein Ar² and Ar³ are eachindependently selected from among phenyl, pyridyl, pyrimidyl,pyridazinyl, pyrrolyl, pyrazinyl, thienyl and furyl.
 9. A pharmaceuticalcomposition, comprising a compound of claim 1 or a pharmaceuticallyacceptable salt of a compound of formula I:

or a pharmaceutically acceptable salt or ester thereof, in apharmaceutically acceptable carrier, wherein: Ar¹ is alkyl, or an arylgroup containing one ring up to three fused rings and from 3 up to about21 members in the ring(s); and A and B are independently selected fromamong pseudohalide, alkyl, alkenyl, alkynyl, aryl, arylalkyl,heteroaryl, thioalkoxy, alkylamino, alkylthio, alkylsulfinyl,alkylsulfonyl, aryloxy, arylamino, arylthio, arylsulfinyl, arylsulfonyl,heteroaryloxy, heteroarylamino, heteroarylthio, haloalkyl, haloaryl,alkoxycarbonyl, alkylcarbonyl, aminocarbonyl, arylcarbonyl, formyl,amido and ureido, in which the alkyl, alkenyl and alkynyl portionscontain from 1 up to about 14 carbon atoms, and are either straight orbranched chains or cyclic, and in which the aryl and heteroaryl portionscontain from 4 to 16 members in the ring.
 10. A method for the treatmentof endothelin-mediated diseases, comprising administering to a subjectin need thereof an effective amount of one or more compounds of formulaI:

or pharmaceutically acceptable salts thereof, wherein: Ar¹ is alkyl, oran aryl group containing one ring up to three fused rings and from 3 upto about 21 members in the ring(s); and A and B are independentlyselected from among halide, pseudohalide, alkyl, alkenyl, alkynyl, aryl,arylalkyl, heteroaryl, alkoxy, thioalkoxy, alkylamino, alkylthio,alkylsulfinyl, alkylsulfonyl, aryloxy, arylamino, arylthio,arylsulfinyl, arylsulfonyl, heteroaryloxy, heteroarylamino,heteroarylthio, haloalkyl, haloaryl, alkoxycarbonyl, alkylcarbonyl,aminocarbonyl, arylcarbonyl, formyl, amido and ureido, in which thealkyl, alkenyl and alkynyl portions contain from 1 up to about 14 carbonatoms, and are either straight or branched chains or cyclic, and inwhich the aryl and heteroaryl portions contain from 4 to 16 members inthe ring; and wherein the effective amount is sufficient to ameliorateone or more of the symptoms of the disease.
 11. The method of claim 10,wherein the disease is selected from the group consisting ofhypertension, cardiovascular disease, asthma, pulmonary hypertension,inflammatory diseases, ophthalmologic disease, menstrual disorders,obstetric conditions, wounds, gastroenteric disease, renal failure,immunosuppressant-mediated renal vasoconstriction,erythropoietin-mediated vasoconstriction, endotoxin shock, anaphylacticshock and hemorrhagic shock.
 12. The method of claim 10, wherein thedisease is selected from the group consisting of asthma and inflammatorydiseases.
 13. A method for inhibiting the binding of an endothelinpeptide to endothelin_(A) (ET_(A)) or endothelin_(B) (ET_(B)) receptors,comprising contacting the receptors an endothelin peptide and with oneor more compounds of formula I:

or pharmaceutically acceptable salts thereof, wherein: Ar¹ is alkyl oran aryl group containing one ring up to three fused rings and from 3 upto about 21 members in the ring(s); and A and B are independentlyselected from among halide, pseudohalide, alkyl, alkenyl, alkynyl, aryl,arylalkyl, heteroaryl, alkoxy, thioalkoxy, alkylamino, alkylthio,alkylsulfinyl, alkylsulfonyl, aryloxy, arylamino, arylthio,arylsulfinyl, arylsulfonyl, heteroaryloxy, heteroarylamino,heteroarylthio, haloalkyl, haloaryl, alkoxycarbonyl, alkylcarbonyl,aminocarbonyl, arylcarbonyl, formyl, amido and ureido, in which thealkyl, alkenyl and alkynyl portions contain from 1 up to about 14 carbonatoms, and are either straight or branched chains or cyclic, and inwhich the aryl and heteroaryl portions contain from 4 to 16 members inthe ring; and wherein: the contacting is effected prior to,simultaneously with or subsequent to contacting the receptors with theendothelin peptide.
 14. A method for altering endothelinreceptor-mediated activity, comprising contacting endothelin receptorswith one or more compounds of formula I:

or pharmaceutically acceptable salts thereof, wherein: Ar₁ is alkyl, oran aryl group containing one ring up to three fused rings and from 3 upto about 21 members in the ring(s); and A and B are independentlyselected from among halide, pseudohalide, alkyl, alkenyl, alkynyl, aryl,arylalkyl, heteroaryl, alkoxy, thioalkoxy, alkylamino, alkylthio,alkylsulfinyl, alkylsulfonyl, aryloxy, arylamino, arylthio,arylsulfinyl, arylsulfonyl, heteroaryloxy, heteroarylamino,heteroarylthio, haloalkyl, haloaryl, alkoxycarbonyl, alkylcarbonyl,aminocarbonyl, arylcarbonyl, formyl, amido and ureido, in which thealkyl, alkenyl and alkynyl portions contain from 1 up to about 14 carbonatoms, and are either straight or branched chains or cyclic, and inwhich the aryl and heteroaryl portions contain from 4 to 16 members inthe ring.
 15. The pharmaceutical composition of claim 9 that isformulated for single dosage administration, and comprises an effectiveamount of one or more of the compounds or pharmaceutically acceptablesalts thereof, wherein the amount is effective for ameliorating thesymptoms of an endothelin-mediated disease.
 16. An article ofmanufacture, comprising packaging material and a compound of formula I:

or a pharmaceutically acceptable salt thereof, wherein: Ar¹ is alkyl, oran aryl group containing one ring up to three fused rings and from 3 upto about 21 members in the ring(s); and A and B are independentlyselected from among halide, pseudohalide, alkyl, alkenyl, alkynyl, aryl,arylalkyl, heteroaryl, alkoxy, thioalkoxy, alkylamino, alkylthio,alkylsulfinyl, alkylsulfonyl, aryloxy, arylamino, arylthio,arylsulfinyl, arylsulfonyl, heteroaryloxy, heteroarylamino,heteroarylthio, haloalkyl, haloaryl, alkoxycarbonyl, alkylcarbonyl,aminocarbonyl, arylcarbonyl, formyl, amido and ureido, in which thealkyl, alkenyl and alkenyl portions contain from 1 up to about 14 carbonatoms, and are either straight or branched chains or cyclic, and inwhich the aryl and heteroaryl portions contain from 4 to 16 members inthe ring, contained within the packaging material, wherein the compoundor salt thereof is effective for antagonizing the effects of endothelin,ameliorating the symptoms of an endothelin-mediated disorder, orinhibiting the binding of an endothelin peptide to an ET receptor withan IC₅₀ of less than about 10 μM; and the packaging material includes alabel that indicates that the compound or salt thereof is used forantagonizing the effects of endothelin, inhibiting the binding ofendothelin to an endothelin receptor or treating an endothelin-mediateddisorder.
 17. The method of claim 10, wherein the disease is glaucoma.